Bispecific antibodies against canine CD3 and CD20

Abstract

The present disclosure provides bispecific canine antigen-binding molecules comprising a first antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD3 and a second antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD20, compositions comprising them, and methods of using them. The present disclosure also provides canine antibodies or antigen-binding portions thereof that bind to canine CD3, compositions comprising them, and methods of using them. The present disclosure also provides canine antibodies or antigen-binding portions thereof that bind to canine CD20, compositions comprising them, and methods of using them.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to UK Patent Application No. 2217993.1 filed November 30, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on November 30, 2023, is named P44660WO1.xml and is 915,096 bytes in size.

[0003] Introduction Cancer is one of the leading causes of death in companion animals. Common cancers include squamous cell carcinoma, breast cancer, prostate cancer, connective tissue cancer, melanoma, oral cancer, pharyngeal cancer, and lymphoma.

[0004] In the human field, cancer immunotherapy has attracted considerable attention over the past 30 years, as several therapeutic approaches have shown successful results. One such approach is the use of bispecific antibodies, which can induce potent effector cells, such as cytotoxic T cells and NK cells, to mediate tumor lysis.

[0005] Cytotoxic T cells, as part of adaptive immunity, are excellent effector cells for mediating killing because they are relatively abundant, capable of proliferating upon activation, and possess potent killing effects. Under physiological conditions, T cells induce cytotoxic activity only against cells expressing major histocompatibility class (MHC) molecules bearing epitopes recognized via the T cell receptor (TCR). The TCR is a protein complex composed of CD3 molecules, typically one CD3εγ heterodimer, one CD3εδ heterodimer, and either an α / β or γ / δ heterodimer associated with two CD3ζ chains. Bispecific T cell engagers can bypass the normal TCR-MHC interaction requirement and trigger T cell activation through one arm that binds to the T cell / CD3 complex, eliciting polyclonal T cell responses against target antigens directed by the second target-recognition arm.

[0006] Since the FDA approval of the anti-CD19 / anti-CD3 bispecific drug blinatumumab, the application of bispecific T cell engagers in humans has increased significantly over the past decade. To date, 43 CD3-based bispecific T cell engager antibodies targeting hematologic and solid tumors are in clinical development (Labrijn et al. Nat Rev Drug Discov. 2021 18:585-608). This class of antibody therapy is considered the next generation of human cancer immunotherapy.

[0007] There remains a need for anti-canine CD3 antibodies.

[0008] The construction of bispecific T cell engager therapeutic molecules in the canine field has been severely limited. Unlike the human field, the canine antibody field has until recently lacked several fundamental technologies necessary to enable complete canine bispecific antibodies, such as methods for heterodimerizing canine heavy chains, methods for limiting light chain mispairing, and purification methods tailored to canine bispecific molecules. Furthermore, the canine field lacks research tools, protocols, and models for T cell biology, including the biology of more specific T cell engager bispecific antibodies. Overcoming these technical challenges would greatly benefit the discovery and development of canine T cell engager bispecific molecules.

[0009] More importantly, such a class of molecules has the potential to fill a critical unmet need in the canine field for both hematological and solid tumors, where, unlike in humans, chemotherapy as the current standard of care does not provide an overall survival benefit.

[0010] Canine lymphoma is one of the most common cancers diagnosed in dogs, accounting for approximately 7-14% of all cancers. As in humans, there are many different types of canine lymphoma, ranging from rapidly progressing cancers to chronic diseases.

[0011] CD20 is a cell surface protein thought to be involved in regulating B cell proliferation and differentiation. This antigen contains four transmembrane domains and is present on the surface of nearly all B cells, both normal and malignant.

[0012] Human antibodies that recognize human CD20, such as rituximab, are used to treat human diseases characterized by an excess number of B cells, or overactive or dysfunctional B cells. These antibodies destroy B cells. Rituximab is considered a revolutionary advance in the treatment of B-cell lymphoma.

[0013] Since the development of antibodies such as rituximab for humans over 20 years ago, several antibodies that recognize canine CD20 have been reported in the literature. However, none are currently in general clinical use. Therefore, there remains a need for alternative therapies. The present invention aims to address this need.

[0014] Beyond anti-canine CD20 monospecific antibody therapy, T-cell engager-based bispecific antibodies may offer significant advantages, leveraging experience in the human field. While rituximab works as a monotherapy or in combination with chemotherapy, there are a large number of relapsed / refractory lymphoma patients in clinical practice. This has led to the discovery and development of second-generation human anti-CD20 monospecific antibody drugs, such as ofatumumab and obinutuzumab, which have significantly enhanced tumor-killing CDC and ADCC activities, respectively, compared to rituximab (Oflazoglu & Audoly 2010 mAbs, 2:14-19). Despite this success, the field is moving toward next-generation anti-CD3 and CD20 bispecific T-cell engagers, while several FDA-approved drugs have entered Phase III clinical trials. Such bispecific molecules induce durable complete responses in patients with relapsed or refractory B-cell lymphoma who have received at least one prior therapy.

[0015] Therefore, the discovery and development of next-generation CD3 / CD20 bispecific antibodies is needed to continue the fight against canine B-cell lymphoma.

[0016] In addition to CD3-based T cell engagers, anti-CD3 activating monospecific antibodies have been widely used in human studies for decades as tools for studying T cell biology. Furthermore, such antibodies have been demonstrated to be effective T cell immunosuppressants in managing rejection after organ transplantation and preventing graft-versus-host disease (Wunderlich M et al. 2014 Blood 123(24):e134-e144). Although highly effective in immunomodulation, the anti-human CD3 drug Orthoclone OKT3 was discontinued due to a severe side effect called cytokine release syndrome (CRS) resulting from strong initial T cell activation. A second-generation design using an effector-function-deficient OKT3 circumvented the problem of high cytokine release, and in 2022 the FDA approved teplizumab for the treatment of patients with recently diagnosed T1D. This approval rekindled interest in this class of therapeutics for modulating immune tolerance.

[0017] These pioneering human studies have paved the way for anti-CD3 therapy to realize its full therapeutic potential. Therefore, the identification of anti-canine CD3 antibodies will benefit the canine field in terms of basic research on T cell biology as well as therapeutic opportunities for canine autoimmune diseases. For example, canine type 1 diabetes is widespread, with approximately 0.2%-1.0% of dogs developing T1D, and this incidence is expected to increase. Summary of the Invention

[0018] In a first aspect, the present invention relates to a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD3. The antibody may comprise three heavy chain variable region (HCVR) complementarity-determining regions (CDRs) and / or three light chain variable region (LCVR) CDRs described herein. The antibody, antigen-binding domain, or antigen-binding portion thereof may comprise the HCVRs and / or LCVRs described herein. The present invention also relates to an immunoconjugate comprising such a canine antibody, antigen-binding domain, or antigen-binding portion thereof, and a pharmaceutical composition comprising such an antibody, antigen-binding domain, or antigen-binding portion thereof. A further aspect relates to the treatment of a disease comprising administering such a canine antibody, antigen-binding domain, or antigen-binding portion thereof. In particular, the disease may be cancer, and also relates to a method for increasing an immune response in a subject, comprising administering such a canine antibody, antigen-binding domain, or antigen-binding portion thereof.

[0019] In another aspect, the invention relates to a bispecific antibody comprising a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD3 and a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to a second canine antigen target.

[0020] In another aspect, the invention relates to a kit comprising a canine antibody, antigen-binding domain, or antigen-binding portion thereof, that binds canine CD3, or a pharmaceutical composition described herein.

[0021] In another aspect, the invention relates to nucleic acid sequences encoding antibodies, antigen-binding domains, or antigen-binding portions thereof, that bind to canine CD3, as described herein.

[0022] In another aspect, the invention relates to a vector comprising a nucleic acid sequence encoding an antibody, antigen-binding domain, or antigen-binding portion thereof, that binds canine CD3, as described herein.

[0023] In another aspect, the invention relates to a host cell comprising such a nucleic acid sequence or vector.

[0024] In another aspect, the invention relates to a method for producing a canine antibody or antigen-binding domain that binds to CD3, the method comprising culturing the isolated host cells and recovering the antibody.

[0025] In another aspect, the present invention provides a method for making a canine antibody or antigen-binding domain that binds to CD3, comprising: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a CD3 antigen; b) generating a library of antibodies from said mice; c) isolating antibodies from said library.

[0026] In another aspect, the invention relates to a method for detecting CD3 protein or an extracellular domain of CD3 protein in a biological sample from a canine subject, the method comprising contacting the biological sample with an antibody, antigen-binding domain, or antigen-binding portion thereof, wherein the antibody, antigen-binding domain, or antigen-binding portion thereof is linked to a detectable label.

[0027] In another aspect, the invention relates to a combination therapy comprising a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD3, or a pharmaceutical composition comprising a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD3.

[0028] In another aspect, the present invention relates to a bispecific canine antigen-binding molecule comprising a first antibody, antigen-binding domain, or antigen-binding portion thereof that specifically binds to canine CD3 and a second antibody, antigen-binding domain, or antigen-binding portion thereof that specifically binds to canine CD20. The first antibody, antigen-binding domain, or antigen-binding portion thereof that specifically binds to canine CD3 can be as described herein. The second antibody, antigen-binding domain, or antigen-binding portion thereof that specifically binds to canine CD20 can be as described herein.

[0029] In another aspect, the present invention relates to a pharmaceutical composition comprising such a bispecific antigen-binding molecule that binds to canine CD3 and canine CD20. The present invention also relates to a method for treating cancer or a B-cell mediated condition in a canine subject in need thereof / a method for increasing an immune response in the subject, comprising administering an effective amount of a bispecific canine antigen-binding molecule.

[0030] The present invention also relates to kits comprising such bispecific canine antigen-binding molecules or pharmaceutical compositions described herein.

[0031] The present invention also relates to nucleic acid sequences encoding such bispecific canine antigen-binding molecules.

[0032] The present invention also relates to a vector comprising such a nucleic acid sequence.

[0033] The present invention also relates to host cells containing such nucleic acid sequences.

[0034] The present invention also relates to a method for producing a bispecific antigen-binding molecule, said method comprising culturing the isolated host cell described herein and recovering said antibody.

[0035] The present invention also relates to a method for detecting CD3 and CD20 proteins in a biological sample from a canine subject, comprising contacting the biological sample with a bispecific antigen-binding molecule described herein, wherein the antibody, antigen-binding domain, or antigen-binding portion thereof is linked to a detectable label.

[0036] The present invention also relates to canine antibodies, antigen-binding domains, or antigen-binding portions thereof that bind to canine CD20. In one embodiment, the canine antibody or antigen-binding fragment portion that binds to canine CD20 is selected from the group consisting of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PM The heavy chain variable region (HCVR) comprises a complementarity determining region (CDR) having an amino acid sequence set forth in Table 4 for X254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268 or PMX269, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto.

[0037] In another aspect, the present invention relates to a pharmaceutical composition comprising the above-described canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD20.

[0038] The present invention also relates to a method for treating cancer or a B-cell mediated condition in a canine subject in need thereof / a method for increasing an immune response in a subject, comprising administering an effective amount of a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD20 as described above.

[0039] The present invention also relates to kits comprising the above-mentioned canine antibody that binds to CD20, antigen-binding domain, or antigen-binding portion thereof, or the above-mentioned pharmaceutical composition.

[0040] The present invention also relates to nucleic acid sequences encoding such canine antibodies, antigen-binding domains, or antigen-binding portions thereof that bind to CD20 as described above.

[0041] The present invention also relates to a vector comprising such a nucleic acid sequence.

[0042] The present invention also relates to host cells containing such nucleic acid sequences.

[0043] The present invention also relates to a method for producing the above-mentioned canine antibody or antigen-binding domain that binds to CD20, which method comprises culturing the above-mentioned isolated host cell and recovering the antibody.

[0044] The present invention also provides a method for producing the above-mentioned canine antibody or antigen-binding domain that binds to CD20, comprising the steps of: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a CD20 antigen; b) generating a library of antibodies from said mice; c) isolating antibodies from said library.

[0045] The present invention also relates to a method for detecting CD20 protein or an extracellular domain of CD20 protein in a biological sample from a canine subject, comprising contacting the biological sample with an antibody, antigen-binding domain, or antigen-binding portion thereof described above, wherein the antibody, antigen-binding domain, or antigen-binding portion thereof is linked to a detectable label.

[0046] The present invention also relates to a method of inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of an antibody, antigen-binding domain, or antigen-binding portion thereof, as described above.

[0047] The present invention also relates to a method for killing tumor cells that express CD20, comprising contacting the cells with the above-described antibody, antigen-binding domain, or pharmaceutical composition, thereby killing the cells that express CD20.

[0048] The invention is further described in the following non-limiting figures. [Brief explanation of the drawings]

[0049] [Figure 1A] Serum titers from CD3-immunized Ky9 mice. Serum antibody titers were measured using flow cytometry after staining HEK cells expressing canine CD3εδ heterodimers on their cell surface. Serum was collected from mice 10 days after the first booster immunization. At this time point, a dose-dependent increase in binding was detected, indicating the presence of specific anti-CD3 antibodies in the serum. [Figure 1B] Serum titers from CD3-immunized Ky9 mice. Serum antibody titers were measured using flow cytometry after staining HEK cells expressing canine CD3εδ heterodimers on their cell surface. Serum was collected from mice 10 days after the second booster immunization. At this time point, a dose-dependent increase in binding was detected, indicating the presence of specific anti-CD3 antibodies in the serum. [Figure 2] Schematic diagram of the four-chain bispecific screening format for anti-CD3 candidates. CD3 candidate sequences were synthesized in a bispecific format for screening. The antibody contains a candidate CD3 arm and an anti-human CD20 arm consisting of rituximab variable sequences. A human Fc containing knob-in-hole mutations was used to enhance heterodimerization. [Figure 3] CD3 cell binding capacity. The graph shows the difference in geometric mean intensity of the signal obtained by flow cytometry of CD3εδ-expressing HEK293 cells or the parental line stained with a candidate CD3 antibody and then stained with a PE-labeled secondary antibody that recognizes human Fc. If the signal obtained when staining CD3εδ-expressing HEK293 cells is higher than the signal obtained when staining wild-type HEK cells, the candidate antibody is considered a binder. [Figure 4A]CD3εδ binding measured by SPR. Exemplary sensorgrams show single-cycle binding kinetics of CD3 candidate antibodies in a bispecific format to canine CD3εδ-Fc: PMX157, PMX158, PMX159, and PMX160. All tested candidate antibodies demonstrated the ability to bind to canine CD3εδ-Fc, albeit to varying degrees. Strains without legends are non-binders that served as negative controls. [Figure 4B] CD3εδ binding measured by SPR. Exemplary sensorgrams show single-cycle binding kinetics of CD3 candidate antibodies in a bispecific format to canine CD3εδ-Fc: PMX161, PMX162, PMX163, and PMX164. All tested candidate antibodies demonstrated the ability to bind to canine CD3εδ-Fc, albeit to varying degrees. Strains without legends are non-binders that served as negative controls. [Figure 5A] Bispecific killing screen. Comparison of bispecific killing activity against CD3 candidates in a bispecific format with IFN-γ release. IFN-γ release after 48 hours of effector:target incubation at an antibody concentration of 1 μg / ml. Effector cells were PBMCs, and target cells were MDCK II cells expressing hCD20+GFP or dCD20+GFP. All IFN-γ curves are baseline-subtracted signals from no-cell controls and plotted as concentrations of IFN-γ released in picograms per milliliter. [Figure 5B] Bispecific killing screen. Comparison of bispecific killing activity and IFN-γ release against CD3 candidates in a bispecific format. A CD3 bispecific antibody with one canine CD3 arm and one rituximab arm (Figure 2) is capable of inducing bispecific killing of canine cells expressing human CD20 at an antibody concentration of 1 μg / ml. Data are plotted as percentage killing, with 100% meaning all cells were killed and 0% meaning the signal was identical to that obtained with control cells (no antibody added). [Figure 6]Dose-response measurements of bispecific killing. Comparison of the ability of a bispecific format CD3 candidate with one canine CD3 arm and one rituximab arm (Figure 2) to induce bispecific killing of canine cells expressing human CD20 using PBMCs with different antibody concentrations. Data are plotted as percentage killing, with 100% meaning all cells were killed and 0% meaning the signal was identical to that obtained with control cells (no antibody added). Also shown on the same plot as the bispecific killing is a representative curve showing IFN-γ release after 48 hours of effector:target incubation with different antibody concentrations. All IFN-γ curves are the signal obtained from the no-cell control minus the baseline and plotted as the concentration of IFN-γ released in picograms per milliliter. [Figure 7] CD3 affinity determination in a bispecific format: The ability of shortlisted candidate CD3 bispecific antibodies (containing one canine CD3 arm and one rituximab arm) to bind to canine CD3εδ-Fc was directly compared (Figure 2), and affinity was measured by SPR. [Figure 8A] Ex vivo T cell activation by monospecific CD3 antibodies. Brightfield images of canine PBMCs cultured for 72 hours on tissue culture plates precoated with the indicated antibodies. The presence of large cell clusters is proportional to the degree of T cell activation. [Figure 8B] Ex vivo T cell activation by monospecific CD3 antibodies. ELISA quantification of IFN-γ in supernatants from canine PBMCs cultured on tissue culture plates precoated with the indicated antibodies. Supernatants were harvested after 3-4 days of culture. The CD3 candidate antibodies tested were able to activate T cells to varying degrees, providing a complete toolset for achieving various levels of T cell activation. [Figure 8C] Ex vivo T cell activation with monospecific CD3 antibodies. Percentage of CD25 and CD5 double positive canine T cells in the presence or absence of PMX159 and costimulatory anti-canine CD28. [Figure 8D]Ex vivo T cell activation with monospecific CD3 antibodies. Percentage of PD-1 and CD5 double positive canine T cells in the presence or absence of PMX159 and costimulatory anti-canine CD28. [Figure 8E] Ex vivo T cell activation with monospecific CD3 antibodies. Percentage of Ki67% positive proliferating CD5+ T cells in the presence or absence of PMX159 and costimulatory anti-canine CD28. [Figure 9] CD20-binding ability of monospecific anti-CD20 candidates. Cell-binding ability of a single dose of candidate monospecific CD20 antibodies at 10 μg / ml. The graph shows the difference in mean fluorescence intensity (MFI) of the signal obtained by flow cytometry of CD20-expressing HEK293 cells stained with candidate CD20 antibodies and then with an FITC-labeled secondary antibody that recognizes canine Fc. All candidates except PMX250 and PMX251 showed strong binding ability to CD20-expressing cells. [Figure 10] Functional screening of monospecific CD20 candidates for CDC capacity. Single dose complement dependent cytotoxicity (CDC) and percentage of cells killed in the assay by each candidate antibody used at 1 μg / ml are shown. [Figure 11] Functional screening of monospecific CD20 candidates for ADCC capacity. Single dose antibody-dependent cellular cytotoxicity (ADCC) assay showing the percentage of cells killed by each candidate antibody used at 0.01 μg / ml. [Figure 12A] The CD3 binding ability of various directly assembled canine CD20 and CD3 heavy chains is compared with that of CD20 or CD3 light chains. Graphic representation of bispecific assemblies composed of either CD3 or CD20 light chains assembled with heavy chain CD3 / CD20 heterodimers using human KiH Fc. [Figure 12B]The CD3-binding ability of various directly assembled canine CD20 and CD3 heavy chains is compared to that of CD20 or CD3 light chains. Cell binding of a single dose of directly assembled CD3 / CD20 bispecific antibody at 1 μg / ml with either CD3 or CD20 VL chains is shown. Images show flow cytometry profiles of CD3εδ-expressing HEK293 cells stained with the directly assembled bispecific antibody followed by a PE-labeled secondary antibody that recognizes human Fc. Secondary staining alone serves as a non-binding control. [Figure 13A] 1. Examination of the compatibility of CD3 and CD20 VL with different CD20 VH. Graphic representation of bispecific assemblies composed of a single CD3 light chain candidate, PMX172VH, and either PMX172VL (CD3VL) or different CD20 VH and VL candidates, using human KiH Fc for heterodimerization. [Figure 13B] Figure 1. Investigation of the compatibility of CD3 and CD20 VL with different CD20 VHs. Figure 2. Cell-based CD3 binding of a single dose of directly assembled CD3 / CD20 bispecific antibody at 1 μg / ml using a single CD3 VL-PMX172VL or cognate CD20 VL, CD3VH-PMX172VH, and different CD20 VHs. Figure 3. Flow cytometry profile of CD3εδ-expressing HEK293 cells stained with the directly assembled bispecific antibody followed by a PE-labeled secondary antibody recognizing human Fc. Secondary staining alone serves as a non-binding control. [Figure 13C] 1. Investigation of the compatibility of CD3 and CD20 VL with different CD20 VH. 2. Cell-based CD20 binding of a single dose of directly assembled CD3 / CD20 bispecific antibody at 1 μg / ml using CD3 VL PMX172VL. 3. Flow cytometry profile of CD20-expressing MDCK cells stained with directly assembled bispecific antibody followed by a PE-labeled secondary antibody recognizing human Fc. Secondary staining alone serves as a non-binding control. [Figure 13D] 13C is a summary of the mean fluorescence intensity of the flow profiles shown in FIG. 13C. [Figure 13E] Investigation of the compatibility of CD3 and CD20 VL with different CD20 VH. Three-chain bispecific assemblies of the VH and VL of the CD3 candidate PMX172, together with the indicated anti-CD20 VH, induce bispecific killing of canine cells expressing human CD20 at different antibody concentrations. Data are plotted as percentage killing, where 100% means that all cells were killed and 0% means that the signal was identical to that obtained with control cells (no antibody added). [Figure 13F] Investigation of the compatibility of CD3 and CD20 VL with different CD20 VH. Percentage of B cell depletion after addition of different concentrations of the three-chain bispecific assembly, as shown in Figure 13E. Data are plotted as percentage of B cell depletion, with 0% meaning that the signal was identical to that obtained with the no antibody control. [Figure 13G] As shown in Figure 13E, the frequency of activated CD8 T cells measured by CD25 activation after addition of different concentrations of the three-chain bispecific assembly. [Figure 14A] Identification of new CD20 VHs for use in bispecific assembly. CD20 binding assay using anti-CD3 PMX172 VH, different VH CD20 binders as indicated, and PMX172 VL at 25 μg / ml, 8.3 μg / ml, 2.8 μg / ml, and 0.92 μg / ml of bispecific candidates. Human KiH was used for Fc-human heterodimerization. The graph shows the mean fluorescence intensity (MFI) of the signal obtained by flow cytometry of CD20-expressing MDCK cells stained with the three-chain bispecific antibody as described, followed by a PE-labeled secondary antibody recognizing human Fc. [Figure 14B] Identification of new CD20 VHs for use in bispecific assembly. Multiple sequence alignment of strong and weak binder anti-CD20 VH sequences with the indicated CDRs. All sequences were aligned to germline V3-5. Gray shading indicates sequence differences from the germline sequences. [Figure 15]Further screening of canine CD20 VHs using a bispecific killing assay. Dose-dependent bispecific cell killing of bispecific assemblies (shown combining the PMX172 common light chain and human KiH Fc for heterodimerization) with anti-CD3 PMX172 VHs and different VH CD20 binders. Target cells: canine cells expressing human CD20. Effectors: canine PBMCs. Data are plotted as percentage killing; 100% means all cells were killed, and 0% means the signal was identical to that obtained with control cells (no antibody added). [Figure 16A] Further screening of canine CD20 VH using an endogenous B cell depletion assay. Percentage of B cell depletion after addition of different concentrations of bispecific assemblies containing anti-CD3 PMX172 VH and different VH CD20 binders, PMX172 VL as a common light chain, and human KiH Fc for heterodimerization. Data are plotted as percentage of B cell depletion; 0% means that the signal was identical to that obtained with the no-antibody control. [Figure 16B] Further screening of canine CD20 VH using an endogenous B cell depletion assay. Percentage of activated CD8 T cells measured by CD25 activation after addition of different concentrations of the bispecific assembly. [Figure 17A] Naive PBMC B cell killing assay of the whole canine CD3 / CD20 bispecific candidate. Percent B cell depletion after addition of 1.25 μg / ml of the whole canine candidate three-chain assembly. Data is plotted as percent B cell depletion, with 0% meaning the signal was identical to that obtained with the no antibody control. [Figure 17B] Naive PBMC B cell killing assay of whole canine CD3 / CD20 bispecific candidate. After addition of 1.25ug / ml of the candidate whole canine three-chain assembly, the percentage of activated CD8 T cells, as measured by CD25 activation, was compared to a no antibody control. [Figure 18A]Whole blood B cell killing assay of the intact canine CD3 / CD20 bispecific candidate. Percent B cell depletion after addition of 1.25 μg / ml of the candidate intact canine three-chain assembly. Data is plotted as percent B cell depletion, with 0% meaning the signal was identical to that obtained with the no antibody control. [Figure 18B] Whole blood B cell killing assay of the complete canine CD3 / CD20 bispecific candidate. After addition of 1.25ug / ml of the candidate complete canine three-chain assembly, the percentage of activated CD8 T cells, as measured by CD25 activation, was compared to a no antibody control. [Figure 19A] 1 is a graphic representation of the canine CD20 knock-in design, which replaces the genomic region encoding all coding exons of mouse CD20 with the corresponding genomic region in canine. [Figure 19B] 1 is a graphic representation of the canine CD3 knock-in (KI) design, in which mouse genomic regions encoding the leader and extracellular domains are replaced with the corresponding genomic regions in canine. [Figure 20] Dose-dependent B cell depletion of CD3 / CD20 bispecific mAb candidates in canine CD3 / CD20 KI mice. Percentage of B cells detected in peripheral blood on day 6 after addition of 0.01, 0.05, 0.1, 0.5, and 1 mg / kg of bispecific candidate. Data are plotted as the percentage of mouse CD19-expressing cells; 0% means the signal is identical to that obtained with the unstained control. Significant differences between sample means were determined using one-way ANOVA. *, P<0.05; **, P<0.01. [Figure 21A]Dose-dependent T cell activation of candidate CD3 / CD20 bispecific mAbs in canine CD3 / CD20 KI mice. Percentage of activated T cells detected in peripheral blood on day 6 after addition of candidate bispecific mAbs at 0.01, 0.05, 0.1, 0.5, and 1 mg / kg. Data are plotted as the percentage of CD8+ T cells expressing activation markers PD1 and TIM3; 0% means the signal is identical to that obtained with the unstained control. Significant differences between sample means were determined using one-way ANOVA. *, P<0.05; **, P<0.01. [Figure 21B] Dose-dependent T cell activation of CD3 / CD20 bispecific mAb candidates in canine CD3 / CD20 KI mice. Correlation plots show the relationship between the percentage of activated CD8+ T cells (CD8+ve, PD1+ve, and TIM3+ve) and the percentage of B cells (CD19+ve). Correlation analysis was performed by Spearman's correlation test, and a p-value <0.05 was considered statistically significant. [Figure 22A] Dose-dependent effector memory activation of CD3 / CD20 bispecific mAb candidates in canine CD3 / CD20 KI mice. Percentage of effector memory T cells detected in peripheral blood on day 6 after addition of 0.01, 0.05, 0.1, 0.5, and 1 mg / kg of candidate bispecific mAb. Data are plotted as the percentage of CD8+ T cells negative for naive T cell markers CD45RA and CD62L. Significant differences between sample means were determined using one-way ANOVA. *, P<0.05; **, P<0.01. [Figure 22B] Dose-dependent effector memory activation of CD3 / CD20 bispecific mAb candidates in canine CD3 / CD20 KI mice. Correlation plots show the relationship between the percentage of effector memory CD8+ T cells (CD8+ve, CD45RA-ve, and CD62L-ve) and the percentage of activated CD8+ T cells (CD8+ve, PD1+ve, and TIM3+ve). Correlation analysis was performed by Spearman's correlation test, and a p-value <0.05 was considered statistically significant. [Figure 23]Cytokine release after bispecific candidate mAb treatment in canine CD3 / CD20 KI mice. Levels of IL-2, IL-6, IFN-γ, and TNF-α were measured from serum samples of mice (n=5) treated with 0.5 mg / kg candidate mAb compared to vehicle control using the LEGENDplex™ Mouse Th Cytokine Bead-based Multiplex Immunoassay. [Figure 24A] Figure 1 shows the time course of B cell depletion in peripheral blood and spleen of canine CD3 / CD20 KI mice with a CD3 / CD20 bispecific mAb candidate. Percentage of B cells identified by CD19-positive staining in the spleen over 21 days after treatment with 0.5 mg / kg of the candidate mAb. Data are plotted as the percentage of positive CD19-expressing cells; 0% means the signal is identical to that obtained with the unstained control. Significant differences between sample means were determined using one-way ANOVA: *, P<0.05; **, P<0.01. [Figure 24B] Figure 1 shows the time course of B cell depletion in peripheral blood and spleen of canine CD3 / CD20 KI mice with CD3 / CD20 bispecific mAb candidates. Percentage of B cells detected in peripheral blood over 21 days after treatment with 0.5 mg / kg of candidate mAbs. Data are plotted as the percentage of positive CD19-expressing cells; 0% means the signal is identical to that obtained with the unstained control. Significant differences between sample means were determined using one-way ANOVA: *, P<0.05; **, P<0.01. [Figure 25A] Time course of cytotoxic T cell activation in peripheral blood and spleen of canine CD3 / CD20 KI mice for a candidate CD3 / CD20 bispecific mAb. Percentage of activated CD8+ T cells identified by PD1+TIM3+ in peripheral blood over 21 days after addition of 0.5 mg / kg of the candidate intact canine three-chain assembly. Data are plotted as the percentage of CD8+ T cells positive for activation markers PD1 and TIM3; 0% means the signal is identical to that obtained with the unstained control. Significant differences between sample means were determined using one-way ANOVA: *, P<0.05; **, P<0.01. [Figure 25B] Figure 1 shows the time course of cytotoxic T cell activation in the peripheral blood and spleen of canine CD3 / CD20 KI mice with a candidate CD3 / CD20 bispecific mAb. Percentage of activated CD8+ T cells detected in the spleen over 21 days after addition of 0.5 mg / kg of the candidate bispecific mAb. Data are plotted as the percentage of CD8+ T cells positive for activation markers PD1 and TIM3; 0% indicates a signal identical to that obtained with the unstained control. Significant differences between sample means were determined using one-way ANOVA. *, P<0.05; **, P<0.01. [Figure 26A] CD8+ T cell counts in peripheral blood and spleen of canine CD3 / CD20 KI mice treated with candidate CD3 / CD20 bispecific mAbs. Percentage of CD8+ T cells detected in the spleen over 21 days after treatment with 0.5 mg / kg of candidate bispecific mAb. Data are plotted as the percentage of positive CD8a-expressing cells; 0% means the signal is identical to that obtained with the unstained control. Significant differences between sample means were determined using one-way ANOVA: *, P<0.05; **, P<0.01. [Figure 26B] CD8+ T cell counts in peripheral blood and spleen of canine CD3 / CD20 KI mice treated with candidate CD3 / CD20 bispecific mAbs. Percentage of CD8+ T cells detected in peripheral blood over 21 days after treatment with 0.5 mg / kg candidate bispecific mAb. Data are plotted as the percentage of positive CD8a-expressing cells; 0% means the signal is identical to that obtained with the unstained control. Significant differences between sample means were determined using one-way ANOVA: *, P<0.05; **, P<0.01. [Figure 27A]Evaluation of canine CD20 expression in A20 tumor cells in a syngeneic mouse model. Cells isolated from tumors generated using canine A20-overexpressing cells and wild-type A20 controls were subcutaneously injected into recipient canine CD3 / CD20 KI mice and stained with PE-canine CD20. Dot plots show side scatter signal plotted against PE signal. Positive PE signal was determined relative to the unstained control. A clear population shift was observed in canine overexpressing tumors compared to wild-type controls, with over 90% being PE-positive. [Figure 27B] Evaluation of canine CD20 expression on A20 tumor cells in a syngeneic mouse model. c Percentage of infiltrating immune cell types present in tumors generated by CD20-overexpressing A20 (n=6). Populations investigated included effector T cells (CD3+ve, CD4+ve), cytotoxic T cells (CD3+ve, CD8a+ve), NK cell markers (CD3-ve, CD49b+ve), PMC-MDSC markers (CD11b+ve, Ly6Clow, Ly6G+ve), and monocytic MDSC markers (CD11b+ve, Ly6Chigh, Ly6G-ve). [Figure 28A] Pharmacokinetics and pharmacodynamics of the canine CD3 and CD20 bispecific candidate in healthy beagles. Percentage of B cells (CD21+ cells) over time relative to the percentage of B cells in peripheral blood before dosing (pre-bleed). Dashed lines correspond to the 24-hour time point after each dosing event (0.01 mg / kg (day 0), 0.05 mg / kg (day 8), and 0.5 mg / kg (day 15)). Statistical analysis was performed in GraphPad Prism, with *=p<0.05 and **=P<0.001 based on one-way ANOVA. [Figure 28B] Pharmacokinetics and pharmacodynamics of the canine CD3 and CD20 bispecific candidate in healthy beagles. Percentage of activated T cells (CD8+, PD1+) detected in peripheral blood 24 hours and 6 days after each administration event of the candidate intact canine three-chain assembly (0.01 mg / kg (day 0), 0.05 mg / kg (day 8), and 0.5 mg / kg (day 15)). [Figure 28C]Pharmacokinetics and pharmacodynamics of the canine CD3 and CD20 bispecific candidate in healthy beagles. Percentage of effector memory T cells (CD45RA-CD62L-) detected in peripheral blood 24 hours and 6 days after each administration event of the candidate intact canine three-chain assembly (0.01 mg / kg (day 0), 0.05 mg / kg (day 8), and 0.5 mg / kg (day 15)). [Figure 29] Combination treatment of CD3 / CD20 bispecific antibodies with chemotherapy and monospecific anti-CD20 therapy. Combination therapy is a successful strategy that maximizes tumor cell elimination while minimizing safety concerns and adverse events. This is evaluated by comparing CHOP, the current standard of care for lymphoma, and what is known in the human field as r-CHOP (CHOP + anti-CD20 monotherapy), with monoclonal therapy alone or in combination (monoclonal CD20 administered to enable B cell debulking, followed by CD3 / CD20 bispecific antibody administration to enable deep tissue penetration). All parameters described in Example 14 were performed as previously described to assess both B cell and T cell population dynamics over time. [Figure 30A] Cell-binding ability of monospecific CD3 mAbs. This graph shows the difference in geometric mean intensity of fluorescent signals obtained by flow cytometry after staining CD3εδ-expressing HEK293 cells or their parental line with a candidate CD3 antibody and then with a PE-labeled secondary antibody that recognizes human Fc. A candidate antibody is considered a binder if the signal obtained when staining CD3εδ-expressing HEK293 cells is higher than the signal obtained when staining wild-type HEK cells. [Figure 30B] Cell-binding ability of monospecific CD3 mAbs. This graph shows the difference in geometric mean intensity of fluorescent signals obtained by flow cytometry after staining CD3εγ-expressing HEK293 cells or their parental line with a candidate CD3 antibody and then with a PE-labeled secondary antibody that recognizes human Fc. A candidate antibody is considered a binder if the signal obtained when staining CD3εγ-expressing HEK293 cells is higher than the signal obtained when staining wild-type HEK cells. DETAILED DESCRIPTION OF THE INVENTION

[0050]

[0023] The following paragraphs further describe various embodiments of the present invention, and each aspect so defined can be combined with any other aspect or aspects, unless expressly indicated to the contrary.

[0051] Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. For example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Duebel, eds., Springer-Verlag, Heidelberg (2010), Handbook of Therapeutic Antibodies, Duebel and Janice M. Reichert, Wiley, (2014).

[0052] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0053] The present inventors have developed fully engineered canine antibodies that specifically bind to canine CD3. These antibodies were generated in transgenic rodents expressing canine V, D, and J genes. Therefore, these antibodies are less likely to be immunogenic when administered to canine subjects than caninized or chimeric antibodies. Furthermore, because these antibodies can be used directly without further modification of the variable regions, there is no risk of reducing affinity or otherwise compromising the antibody. Other techniques risk development or efficacy problems due to ex vivo combination of canine-derived antibody sequences with antibody sequences from other species, typically rodents. Thus, the present invention relates to canine antibodies or antigen-binding portions thereof that bind to canine CD3.

[0054] The present invention further relates to bispecific canine antigen-binding molecules comprising a first antibody, antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD3, and a second antibody, antigen-binding domain or antigen-binding portion thereof that specifically binds to another canine antigen, for example CD20.

[0055] The properties of the antibodies, antigen-binding domains, and antigen-binding portions thereof of the present invention can be exploited in the pharmaceutical formulations, as well as in the therapeutic methods and uses described herein.

[0056] The term CD3 refers to antigenic cluster of differentiation 3. CD3 is a multimeric protein complex, historically known as the T3 complex, and is composed of four distinct polypeptide chains: epsilon (ε), gamma (γ), delta (δ), and zeta (ζ), which assemble into three pairs of dimers (εγ, εδ, ζζ). The CD3 complex functions as a T cell coreceptor, non-covalently binding to the T cell receptor (TCR). Unless otherwise specified, the term CD3 used herein refers to canine CD3.

[0057] The antibodies, antigen-binding domains, and antigen-binding portions thereof specifically bind to wild-type canine CD3, particularly CD3εδ dimers. The nucleic acid and amino acid sequences of wild-type canine CD3 subunits are shown in Table 1. The amino acid sequence of wild-type CD3ε is SEQ ID NO: 4, and the amino acid sequence of wild-type CD3δ is SEQ ID NO: 5. Unless otherwise specified, the term CD3 as used herein refers to canine CD3.

[0058] The terms "CD3 antigen-binding domain," "CD3 binding molecule / protein / polypeptide / agent / moiety," "CD3 antigen-binding molecule / protein / polypeptide / agent / moiety," "anti-CD3 antibody," and "anti-CD3 antibody or antigen-binding portion thereof" all refer to a molecule capable of specifically binding to canine CD3 antigen. Binding can be demonstrated by standard methods, for example, by reference to a negative control test using an antibody of irrelevant specificity.

[0059] The term CD20 refers to the B lymphocyte antigen CD20. The antibodies, antigen-binding domains, and antigen-binding portions thereof are defined in SEQ ID NO: 22 (nucleotide sequence) and SEQ ID NO: 24 (amino acid sequence) and specifically bind to wild-type canine CD20 as shown in Table 1. Unless otherwise specified, the term CD20 as used herein refers to canine CD20. The B lymphocyte antigen CD20 or CD20 is expressed on the surface of all B cells, starting from the pro-B phase (CD45R+, CD117+) and gradually increasing in concentration until maturity. In humans and dogs, CD20 is encoded by the MS4A1 gene.

[0060] The terms "CD20 antigen-binding domain," "CD20 binding molecule / protein / polypeptide / agent / moiety," "CD20 antigen-binding molecule / protein / polypeptide / agent / moiety," "anti-CD20 antibody," and "anti-CD20 antibody or antigen-binding portion thereof" all refer to a molecule capable of specifically binding to canine CD20 antigen. Binding can be demonstrated by standard methods, for example, by reference to a negative control test using an antibody of irrelevant specificity.

[0061] The antibodies, antigen-binding domains, or antigen-binding portions thereof of the invention (including multispecific, e.g., bispecific or trispecific binding agents as described herein that "bind" or are "capable of binding" to an antigen of interest, i.e., canine CD3, CD3 and CD20, or CD20) are those that bind to the antigen with sufficient affinity such that the antibodies, antigen-binding domains, or antigen-binding portions thereof are useful as therapeutic agents that target cells or tissues expressing the respective antigens described herein.

[0062] The antibodies, antigen-binding domains, or antigen-binding portions thereof described herein specifically bind to canine CD3. In other words, binding to the canine CD3 antigen is measurably different from non-specific interactions. In particular, the antibodies described herein do not cross-react with mouse CD3.

[0063] Also described are antibodies, antigen-binding domains, or antigen-binding portions thereof that specifically bind to canine CD20. In other words, binding to the canine CD20 antigen is measurably different from non-specific interactions. In particular, the antibodies described herein do not cross-react with mouse CD20.

[0064] As used herein, the terms "specific binding" or "specifically binds" or "is specific for" a particular polypeptide or epitope on a particular polypeptide target means, for example, at least about 10 -6 M, alternatively at least about 10 -7 M, alternatively at least about 10 -8 M, alternatively at least about 10 -9M, alternatively at least about 10 -10 M, alternatively at least about 10 -11 M, alternatively at least about 10 -12 In one embodiment, the KD is at least about 10 -8 M ~ about 10 -9 M, e.g., in one embodiment, KD is in the nanomolar range. In one embodiment, the term "specific binding" refers to binding of a molecule to a particular polypeptide or an epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. KD and K D The terms are used interchangeably herein. Additional binding affinities are described elsewhere herein.

[0065] As used herein, the term "antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising at least one complementarity-determining region (CDR), alone or in combination with one or more additional CDRs and / or framework regions (FRs), that specifically binds to a particular antigen. In certain embodiments, the antigen-binding molecule is an antibody or a portion of an antibody, as these terms are defined elsewhere herein. In some embodiments, the antigen-binding domain specifically binds to the canine CD3 antigen. In some embodiments, the antigen-binding domain specifically binds to the canine CD20 antigen. The term "antigen-binding molecule" includes antibodies and antigen-binding portions of antibodies, including, for example, bispecific antibodies.

[0066] As used herein, the term "bispecific antigen-binding molecule" refers to a protein, polypeptide, or molecular complex comprising at least a "first antigen-binding domain" and a "second antigen-binding domain." Each antigen-binding domain in a bispecific antigen-binding molecule comprises at least one CDR that specifically binds to a particular antigen, either alone or in combination with one or more additional CDRs and / or FRs. In the context of the present invention, the first antigen-binding domain specifically binds to a first different antigen (e.g., canine CD3), and the second antigen-binding domain specifically binds to a second different antigen (e.g., canine CD20).

[0067] As used herein, the term "antibody" refers broadly to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or an antigen-binding portion thereof, or any functional fragment, mutant, variant, or derivative thereof that retains the essential epitope-binding properties of an Ig molecule.

[0068] In a full-length antibody, each heavy chain is composed of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. H 1. C H 2 and C H Each light chain is composed of three domains: a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is composed of one domain, C L It consists of:

[0069] Heavy and light chain variable regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable region is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0070] Immunoglobulin molecules generally can be of any isotype, class, or subclass. H The three domains are C of canine IgG subtypes, e.g., IgG-A, IgG-B, IgG-C, and IgG-D. H There are three domains.

[0071] Dogs have four IgG heavy chains, designated A, B, C, and D. These heavy chains represent four distinct subclasses of canine IgG, designated IgG-A, IgG-B, IgG-C, and IgG-D. The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. (Vet. Immunol. Immunopathol. 80:259-270 (2001)). The amino acid and DNA sequences of these heavy chains are also available from the GenBank database (IgGA: Accession No. AAL35301.1, IgGB: Accession No. AAL35302.1, IgGC: Accession No. AAL35303.1, IgGD: Accession No. AAL35304.1). Canine antibodies also contain two types of light chains: kappa and lambda (GenBank Accession Number: Kappa light chain amino acid sequence ABY 57289.1, GenBank Accession Number ABY 55569.1). The antibodies herein may have a lambda light chain or a kappa light chain. In one embodiment, the light chain is a lambda light chain.

[0072] The term "CDR" refers to a complementarity-determining region within an antibody variable sequence. Each heavy and light chain variable region has three CDRs, designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs can be defined differently according to various systems known in the art.

[0073] The Kabat complementarity-determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The Kabat numbering system is typically used to refer to residues within the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain). Another system is the ImMunoGeneTics (IMGT) numbering scheme (Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005)). With the advent of large-scale single-cell VDJ sequencing data, enclone, a system for calculating clonotypes from single-cell data, has become a useful tool for rational analysis (Jaffe et al., enclone: ​​precision clonotyping and analysis of immune receptors. bioRxiv July 9, 2022. https: / / doi.org / 10.1101 / 2022.04.21.489084). enclone is a computational tool available at https: / / 10xgenomics.github.io / enclone / that employs the Adaptive Immune Receptor Repertoire (AIRR) numbering and CDR definitions (Heiden et al. front Immunol. 2018;9:2206) and will be used herein for numbering and CDR definitions unless otherwise specified.

[0074] A chimeric antibody is a recombinant protein that contains variable domains comprising the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent or human antibody, while the constant domains of the antibody molecule are derived from the constant domains of a canine antibody.

[0075] As used herein, the term "caninized antibody" refers to a form of recombinant antibody that contains both canine and non-canine (e.g., murine) antibody sequences. Generally, caninized antibodies contain substantially all of at least one or more, typically two, variable domains, in which all or substantially all of the hypervariable loops of the variable domains correspond to those of a non-canine immunoglobulin, and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining framework) are variable domains of a canine immunoglobulin sequence. A caninized antibody may contain both three heavy chain CDRs and three light chain CDRs derived from a murine or human antibody, together with a canine frame or a modified canine frame. The modified canine frame contains one or more amino acid changes that can further optimize the effectiveness of the caninized antibody, for example, to increase binding to its target. For example, the non-canine sequences of the hypervariable loops can be further compared to the canine sequences, and many residues can be altered to resemble the authentic canine sequences as closely as possible.

[0076] In contrast, preferred complete canine antibodies according to the present invention have canine variable regions and do not contain complete or partial CDRs or FRs from another species. Advantageously, the complete canine antibodies described herein are obtained from transgenic mice containing canine immunoglobulin sequences. Antibodies produced in these immunized mice are developed through in vivo B cell signaling and development, allowing natural affinity maturation, including in vivo V(D)J recombination, in vivo ligation-associated diversification, in vivo heavy and light chain pairing, and in vivo hypermutation. Complete canine antibodies produced in this manner generate antibodies with optimal properties for developability, minimizing lengthy lead optimization prior to large-scale manufacturing. Advantageously, such complete canine antibodies present the lowest possible risk of immunogenicity when introduced into patient animals, thereby facilitating repeated dosing regimens. Given that ex vivo mAb engineering carries the risk of introducing developmental defects, immunogenicity, and reduced affinity (as outlined above), the complete canine antibodies of the present invention are therefore most likely to be effective therapies in clinical settings. Thus, in one embodiment, the term canine antibody refers to a complete canine antibody.

[0077] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation, carbohydrate addition). Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen.

[0078] The term "antigen-binding site" refers to the portion of an antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen-binding site may be provided by one or more antibody variable domains. An antigen-binding site is usually located within the relevant V domains of an antibody or antibody fragment. H and V L Included within.

[0079] The term "epitope" or "antigenic determinant" refers to a site on the surface of an antigen to which an immunoglobulin, antibody, or antibody fragment specifically binds. Generally, an antigen has several or many different epitopes, reacting with many different antibodies. This term specifically includes linear and conformational epitopes. Epitopes within protein antigens can be formed both from contiguous amino acids (usually linear epitopes) or from noncontiguous amino acids juxtaposed by tertiary folding of the protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are usually, but not always, retained by exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost by treatment with denaturing solvents. Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining the epitope bound by a given antibody or antibody fragment (i.e., epitope mapping by alanine scanning mutagenesis or pepscan) are well known in the art and include, for example, immunoblot assays and immunoprecipitation assays in which overlapping or consecutive peptides are tested for reactivity with a given antibody or antibody fragment. If two antibodies recognize the same or sterically overlapping epitopes, the antibodies bind to "essentially the same epitope" as a reference antibody. The most widely used rapid method for determining whether two epitopes bind to the same or sterically overlapping epitopes is a competition assay, which can be configured in a variety of formats using either labeled antigen or labeled antibody.

[0080] In one embodiment, the epitope can be determined by site-directed mutagenesis, for example, using alanine scanning. In one embodiment, the epitope is a linear epitope. In one embodiment, the epitope is a conformational epitope.

[0081] The present invention also relates to antibodies, antigen-binding domains, or antigen-binding portions thereof that compete with the antibodies, antigen-binding domains, or antigen-binding portions thereof according to the invention.

[0082] Proteolytic digestion of antibodies releases different fragments called Fv (fragment variable), Fab (fragment antigen binding), and Fc (fragment crystallization). The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The constant domains of the Fc fragment are responsible for mediating the effector functions of the antibody.

[0083] The present invention extends to antigen-binding portions or antigen-binding fragments of antibodies. The terms "binding portion" and "fragment" are used interchangeably herein. An antibody fragment / portion is a portion of an antibody, such as F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy chain (V), or the like. H ), variable light chain (V L ) domains. Functional fragments of full-length antibodies retain the target specificity of the entire antibody. Therefore, recombinant functional antibody fragments such as Fab (fragment antibody), scFv (single-chain variable fragment), and single-domain antibodies (dAb) have been used in the development of therapeutics as an alternative to mAb-based therapeutics.

[0084] The present invention also extends to antibody mimetics comprising the sequences of the invention.

[0085] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chain) that contribute amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0086] "Single-chain Fv", also abbreviated as "sFv" or "scFv", consists of two variable domains V connected to a single polypeptide chain. H and V L The antibody fragment consisting of scFv fragment (approximately 25 kDa) is H Domain and V L The domains tend to associate and dissociate non-covalently through hydrophobic interactions, although stable fragments can be engineered by linking the domains with flexible hydrophilic linkers to create single-chain Fvs (scFvs).

[0087] The smallest antigen-binding fragment is a single variable fragment, i.e., a variable heavy chain (V H ) or variable light chain (V L ) domain. V H Domain and V L Each domain is capable of binding to an antigen. Target binding does not require binding to a respective light / heavy chain partner, or indeed the presence of other parts of the entire antibody. The antigen-binding entity of an antibody consists of one single domain (V H or V L These antibodies are generally called "single domain antibodies" or "immunoglobulin single variable domains." Therefore, single domain antibodies (approximately 12-15 kDa) are characterized by a V H Domain or V L The term "dAb," for "domain antibody," generally refers to a single immunoglobulin variable domain (V) that specifically binds to an antigen. H , V HH , or V L ) polypeptide.

[0088] The antibodies, antigen-binding domains and antigen-binding portions or fragments thereof according to the invention are preferably isolated.

[0089] The term "isolated" refers to a moiety that is isolated from its natural environment. For example, the term "isolated" refers to an antibody or fragment thereof that is substantially free of other antibodies, antibodies, or antibody fragments. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0090] As used herein, the term "homology" or "identity" generally refers to the percentage of amino acid residues in a sequence that are identical to the residues of a reference polypeptide to which the sequence is compared, after aligning the sequences and, in some embodiments, introducing gaps as necessary to achieve the maximum percent homology, and without considering conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N-terminal nor C-terminal extensions, tags, or insertions are considered to reduce identity or homology. Methods and computer programs for alignment are well known. The percent identity between two amino acid sequences can be determined using well-known mathematical algorithms.

[0091] As used herein, "amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analogue that may be present at a particular, defined position. Amino acid encompasses both naturally occurring and synthetic amino acids, although in most cases, only naturally occurring amino acids are used when proteins are recombinantly produced.

[0092] As used herein, "substitution of an amino acid residue" with another amino acid residue in the amino acid sequence of a protein or polypeptide (e.g., an antibody) described herein is equivalent to "replacing an amino acid residue" with another amino acid residue and indicates that a particular amino acid residue at a particular position in the original (e.g., wild-type / germline) amino acid sequence has been replaced (or substituted) with a different amino acid residue. This can be done using standard techniques available to those of skill in the art, such as recombinant DNA technology. Amino acids are altered compared to the native (wild-type / germline) sequence as found in nature in wild-type (wt), but can also be generated in IgG molecules that contain other changes compared to the native sequence. "Wild-type" or "WT" or "native" herein refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, or immunoglobulin has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0093] Canine antibodies that bind to canine CD3εδ or antigen-binding portions thereof In one aspect, the present invention relates to canine antibodies, antigen-binding domains or antigen-binding portions thereof that bind to canine CD3, in particular CD3εδ.

[0094] Canine antibodies, antigen-binding domains or antigen-binding portions thereof that bind to canine CD3, particularly CD3εδ, according to the present invention include: a) specifically binds to canine CD3, particularly CD3εδ; b) activating the canine T cell receptor as demonstrated by agonistic binding to canine CD3, particularly CD3εδ, and mediating cell killing in a bispecific format where one arm recognizes CD3, particularly CD3εδ, and the other arm recognizes CD20, as shown, for example, in Example 4; c) activating canine T cell receptors, resulting in ex vivo or in vivo T cell activation, as determined, for example, by the concentration of interferon gamma, IL-2 or other T cell cytokines secreted upon activation, as shown in the Examples; d) activating canine T cells, resulting in upregulation of surface marker expression such as CD25, CD69, PD-1 and / or other known markers for T cell activation, as shown in the Examples; e) activating canine T cells and inducing morphological changes such as T cell and / or PBMC clustering, as shown in the Examples; and / or f) have one or more of the following properties: activate canine T cells and induce T cell proliferation as demonstrated by markers such as Ki67, as shown in the Examples.

[0095] One potential use of the agonist anti-CD3 antibodies, antigen-binding domains, or portions thereof of the present invention is the ex vivo activation and expansion of canine T cells, which can be achieved using anti-CD3 antibodies alone, in combination with anti-CD28 antibodies and / or other T cell stimulators (such as, for example, IL-2).

[0096] Another potential application of agonistic monospecific anti-CD3 antibodies or antigen-binding domains is T cell immune tolerance in the treatment of autoimmune diseases such as T1D and host-graft rejection disorders. For this application, canine Fc lacking effector function is used. Monospecific anti-CD3 antibodies or antigen-binding domains may be able to induce T cell anergy. T cell anergy is a state of prolonged hyporesponsiveness / unresponsiveness. It is induced by stimulating T cells via the TCR in the absence of costimulatory signals such as CD28. Inducing T cell anergy results in an immunosuppressive state and can be used to treat autoimmune diseases such as type 1 diabetes. Measures of T cell activation are known in the art and include surface markers, T cell proliferation, and cytokine release.

[0097] In one embodiment, a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD3 according to the present invention is a) binds to canine CD3, particularly CD3εδ, with a binding dissociation equilibrium constant (KD) of 100 nM to 1000 nM; b) agonistically bind to canine CD3, particularly CD3εδ, activate the canine T cell receptor, and mediate target-specific cell killing in vitro and ex vivo in a bispecific format, where one arm recognizes CD3, particularly CD3εδ, and the other arm recognizes CD20, as shown, for example, in Example 4; c) eliciting T cell surface upregulation of markers such as CD25, CD69, etc. upon target-mediated cell killing, e.g., in a bispecific format where one arm recognizes CD3, particularly CD3εδ, and the other arm recognizes a target antigen such as CD20 in vitro, ex vivo, and in vivo, as demonstrated in the Examples; d) activating canine T cells in vitro to reduce, e.g., minimize, the secretion of cytokines such as IFN-γ; and / or e) A bispecific format in which one arm recognizes CD3, particularly CD3εδ, and the other arm recognizes, for example, CD20, and which mediates target-specific cell killing in vivo with minimal cytokine release, such as release of IFN-γ, IL-2, IL-6 and / or TNF-α.

[0098] Antibodies, antigen-binding domains, or antigen-binding portions thereof, exhibiting properties a) through e) in the preceding paragraphs are particularly useful for use in bispecific antibody molecules having an anti-CD3 arm for binding to T cells and a target cell-specific arm directed to antigens such as CD20, CD19, BCMA, CD123, CD33, and CD38.

[0099] In yet another embodiment, the canine antibody, antigen-binding domain, or antigen-binding portion thereof binds to canine CD3, particularly CD3εδ, with a monovalent binding dissociation equilibrium constant (KD) of less than 100 nM or between 100 nM and 1 μM. Antibodies, antigen-binding domains, or antigen-binding portions thereof that exhibit such kinetic properties are particularly useful in monovalent formats and can be used as agonist antibodies to activate T cells.

[0100] These properties described above can be measured by methods known in the art, such as those disclosed in the Examples, including in vivo studies in mouse models or dogs.

[0101] In one embodiment, a canine antibody, antigen-binding domain, or antigen-binding fragment portion that binds to canine CD3 comprises a complementarity determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. Complementarity determining region (CDR) refers to the three CDRs, i.e., CDR1, 2, and 3.

[0102] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment thereof comprises (a) a complementarity determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto, and (b) a CDR of a light chain variable region (LCVR) having an amino acid sequence set forth in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In other words, in one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment thereof comprises (a) an HCVR CDR set forth for one of the PMX molecules in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto, and (b) an LCVR CDR set forth for one of the PMX molecules in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment thereof is selected from the group consisting of PMX157, PMX158, PMX160, PMX190, PMX162, PMX163, PMX189, PMX165, PMX167, PMX168, PMX169, PMX170, PMX171, PMX172, PMX173, PMX174, PMX175, PMX176, PMX177, PMX178, PMX179, PMX180, PMX181, PMX182, PMX183, PMX184, PMX185, PMX186, PMX187, PMX188, PMX189, PMX190, PMX191, PMX192, PMX193, PMX194, PMX195, PMX196, PMX197, PMX198, PMX199, PMX200, PMX201, PMX202, PMX203, PMX204, PMX205, PMX206, PMX207, PMX208, PMX209, PMX300, PMX310, PMX311, PMX312, PMX313, PMX314, PMX315, PMX316, PMX317, PMX318, PMX319, PMX409, PMX410, PMX420, PMX421, PMX422, PMX423, PMX424, PMX425, PMX426, PMX427, PMX428, PMX429, PMX500, PMX511, PMX512, PMX513, PMX514, PMX515, PMX51 Comprising the HCVR CDRs and LCVR CDRs of PMX178, PMX179, PMX180, PMX181, PMX182, PMX183, PMX184, PMX185, PMX186, PMX187, PMX188, PMX190, PMX192, PMX193, PMX194, PMX195, PMX196, PMX197, PMX198, PMX200, PMX272, PMX273, PMX285, or PMX286.

[0103] In one embodiment, the present invention relates to an isolated canine antibody, antigen-binding domain, or antigen-binding portion thereof, that binds to canine CD3, said antibody comprising: a) an HCVR CDR1 sequence comprising or consisting of a SEQ ID NO: set out for a PMX molecule in Table 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto; b) an HCVR CDR2 sequence comprising or consisting of a SEQ ID NO: set out for each PMX molecule of Table 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto; c) an HCVR CDR3 sequence comprising or consisting of a SEQ ID NO: set out for each PMX molecule of Table 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto; d) an LCVR CDR1 sequence comprising or consisting of a SEQ ID NO: set out for each PMX molecule of Table 2, or an amino acid sequence having at least 60%, 70%, 80% or 90% sequence identity thereto; e) an LCVR CDR2 sequence comprising or consisting of a SEQ ID NO: set out for each PMX molecule in Table 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto; and f) A LCVR CDR3 sequence comprising or consisting of the SEQ ID NO: set out for the respective PMX molecule in Table 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto.

[0104] In one embodiment, an isolated canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD3, wherein said antibody, antigen-binding domain, or antigen-binding portion thereof is: a) an HCVR CDR1 sequence comprising or consisting of a SEQ ID NO: shown for a PMX molecule in Table 2; b) an HCVR CDR2 sequence comprising or consisting of the SEQ ID NO: shown for each PMX molecule in Table 2; c) an HCVR CDR3 sequence comprising or consisting of the SEQ ID NO: set out for each PMX molecule in Table 2; d) an LCVR CDR1 sequence comprising or consisting of the SEQ ID NO: shown for each PMX molecule in Table 2; e) an LCVR CDR2 sequence comprising or consisting of the SEQ ID NO shown for each PMX molecule in Table 2, and f) comprising an LCVR CDR3 sequence comprising or consisting of a SEQ ID NO: set out for the respective PMX molecule of Table 2, or an isolated canine antibody, antigen-binding domain, or antigen-binding portion thereof, having the above CDRs, but having one or more CDRs with 1, 2, 3, 4, or 5 amino acid substitutions in one or more LCVR or HCVR CDRs compared to a reference CDR sequence.

[0105] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment portion thereof comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto, and (b) a light chain variable region (LCVR) having an amino acid sequence set forth in Table 2, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment portion thereof comprises the HC CDRs and LC CDRs of a PMX molecule set forth in Table 2.

[0106] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment thereof is selected from the group consisting of PMX157, PMX158, PMX160, PMX190, PMX162, PMX163, PMX189, PMX165, PMX167, PMX168, PMX169, PMX170, PMX171, PMX172, PMX173, PMX174, PMX175, PMX176, PMX177, PMX178, PMX179, PMX180, PMX181, PMX182, PMX183, PMX184, PMX185, PMX186, PMX187, PMX188, PMX189, PMX190, PMX191, PMX192, PMX193, PMX194, PMX195, PMX196, PMX197, PMX198, PMX199, PMX200, PMX201, PMX202, PMX203, PMX204, PMX205, PMX206, PMX207, PMX208, PMX209, PMX300, PMX310, PMX311, PMX312, PMX313, PMX314, PMX315, PMX316, PMX317, PMX318, PMX319, PMX401, PMX402, PMX403, PMX404, PMX405, PMX406, PMX407, PMX408, PMX409, PMX501, PMX510, PMX511, PMX512, PMX513, PMX514, PMX515, PMX516, PMX517, PMX51 8, PMX179, PMX180, PMX181, PMX182, PMX183, PMX184, PMX185, PMX186, PMX187, PMX188, PMX190, PMX192, PMX193, PMX194, PMX195, PMX196, PMX197, PMX198, PMX200, PMX272, PMX273, PMX285, or PMX286.

[0107] In one embodiment, the antibody, antigen-binding domain, or antigen-binding portion thereof, a) an HCVR sequence comprising or consisting of a sequence of a SEQ ID NO: set forth for a PMX molecule in Table 2, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid alterations (e.g., deletions, additions or substitutions) in the HCVR framework regions compared to a reference HCVR; and b) An LCVR sequence comprising or consisting of the sequence of the SEQ ID NO shown for each PMX molecule in Table 2, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications (e.g., deletions, additions or substitutions) in the LCVR framework regions compared to a reference LCVR.

[0108] In one embodiment, the antigen-binding domain or antigen-binding portion thereof can be a F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy chain (V H ) domain, or the variable light chain (V L )

[0109] In one embodiment, the antigen-binding domain or antigen-binding portion is a heavy chain and comprises the HCVR CDRs set forth for a PMX molecule shown in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, the canine antigen-binding domain or antigen-binding fragment portion comprises or consists of the HCVR CDRs set forth for PMX160, PMX162, PMX169, PMX170, PMX171, PMX172, PMX186, PMX187, PMX190, PMX188, or PMX189.

[0110] In one embodiment, the antigen-binding domain or antigen-binding portion comprises or consists of a heavy chain variable region and comprises an HCVR set forth for a PMX molecule shown in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto. Thus, in one embodiment, the canine antigen-binding domain or antigen-binding fragment portion comprises or consists of an HCVR having an amino acid sequence shown in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto.

[0111] In one embodiment, the canine antigen-binding domain or antigen-binding fragment portion comprises or consists of the HCVRs set forth for PMX160, PMX162, PMX169, PMX170, PMX171, PMX172, PMX186, PMX187, PMX190, PMX188, or PMX189.

[0112] In one embodiment, the above-described antibody, antigen-binding domain, or antigen-binding portion thereof comprises an Fc region, eg, a canine Fc region, eg, a canine IgGB Fc region.

[0113] In one embodiment, the canine antibody or antigen-binding portion described herein specifically binds CD3 and comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 48, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 49, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 50, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 52. In another embodiment, the canine antibody or antigen-binding portion described herein specifically binds CD3 and comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 87, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 88, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 89, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 90, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 91, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 92. In yet another embodiment, the canine antibody or antigen-binding portion described herein specifically binds CD3 and comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 127, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 128, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 129, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 130, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 131, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 132. In one embodiment, the canine antibody or antigen-binding portion described herein specifically binds CD3 and comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 167, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 168, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 169, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 170, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 171, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 172.

[0114] In one embodiment, the canine antibodies or antigen-binding portions described herein specifically bind to CD3 and comprise a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 46. In another embodiment, the canine antibodies or antigen-binding portions described herein specifically bind to CD3 and comprise a VH comprising the amino acid sequence of SEQ ID NO: 84 and a VL comprising the amino acid sequence of SEQ ID NO: 86. In yet another embodiment, the canine antibodies or antigen-binding portions described herein specifically bind to CD3 and comprise a VH comprising the amino acid sequence of SEQ ID NO: 124 and a VL comprising the amino acid sequence of SEQ ID NO: 126. In one embodiment, the canine antibodies or antigen-binding portions described herein specifically bind to CD3 and comprise a VH comprising the amino acid sequence of SEQ ID NO: 164 and a VL comprising the amino acid sequence of SEQ ID NO: 166.

[0115] The variable region sequences described herein, including but not limited to the amino acid and nucleotide sequences (and / or fragments thereof) shown in Table 2, can be used in combination with one or more amino acid and / or nucleotide sequences encoding one or more constant chains (and / or fragments thereof) of an antibody molecule. For example, the variable region amino acid sequences shown in Table 2 can be linked to the constant region of any antibody molecule from the same or a different species (e.g., human, goat, rat, sheep, chicken) from which the variable region amino acid sequence was derived. Preferably, the variable region amino acid sequences shown in Table 2 are linked to the constant region of a canine antibody, which may be the constant region of any of canine IgG A, B, C, or D. In one embodiment, the constant region is the canine IgG B constant region. The constant regions of canine IGGB (SEQ ID NO: 28), canine IGK, or canine IGLC5 may be used. Variants of constant regions with altered effector regions, such as a variant of canine IGGB (SEQ ID NO: 30), can also be used. These variants can be generated by introducing mutations into canine IgG-B that abolish effector function. Canine IgG-B can be modified to reduce or abolish the effector function of canine IgG-B when compared to the same polypeptide comprising a wild-type IgG-B Fc domain. Regions of the amino acid sequence of the Fc domain that can be modified include the lower hinge, proline region, and SHED region, where interactions with FcgammaR and C1q may occur. Examples of such mutations are described in WO2023 / 012486, which is incorporated herein by reference.

[0116] Other such variants may include charge pair combinations in the canine CH3 domain, which may significantly enhance heavy chain heterodimerization over homodimer formation. This can minimize the formation of homodimer contaminants in the production of bispecific antibodies. These charge pair combinations may be present within the canine IgG CH3 domain interface of the Fc region, where the IgG is selected from IgG-A, B, C, or D. Both the first canine IgG CH3 domain and the second canine IgG CH3 domain may be complementarily designed so that each CH3 domain (or polypeptide containing it) does not substantially homodimerize with itself or homodimerizes at a slower rate, but is forced to heterodimerize with the other complementarily designed CH3 domain. In other words, the first and second CH3 domains may heterodimerize, and almost no homodimers are formed between the two first or second CH3 domains. Examples of such mutations are described in WO2021 / 214460 A1, incorporated herein by reference.

[0117] Variants may also contain mutations in the canine CH2 or CH3 IgG Fc domain that result in differential affinity for binding affinity reagents and / or improved stability. For example, a binding molecule may have differential affinity for binding to Protein A compared to a wild-type IgG Fc domain. Differential affinities of immunoglobulin heavy chains allow for optimal isolation of the binding protein or antibody. For example, a suitable variant IgG Fc domain may contain one or more amino acid substitutions that increase the affinity for binding to Protein A or one or more amino acid substitutions that decrease the affinity for binding to Protein A. Examples of such mutations are described in GB2311984.5, which is incorporated herein by reference.

[0118] Thus, in one embodiment, the antigen-binding domain, or antibody, or antigen-binding portion thereof, comprises an Fc-binding-deficient variant resulting from mutation of canine IgG-B. In another embodiment, the antibody, or antigen-binding portion thereof, comprises an enhanced heavy chain heterodimerization variant resulting from mutation of the canine CH3 domain of canine IgG-A, B, C, or D. In another embodiment, the antigen-binding domain, or antibody, or antigen-binding portion thereof, comprises a variant with mutations in the canine CH2 or CH3 IgG Fc domain that result in differential affinity to binding affinity reagents and / or improved stability. The antigen-binding domain, or antibody, or antigen-binding portion thereof, may have a single mutation that results in a single mutant phenotype (e.g., reduced Fc effector function) or may have multiple mutations that result in multiple phenotypes (e.g., reduced Fc effector function, enhanced heavy chain heterodimerization, and altered Protein A binding affinity).

[0119] Also included within the scope of the present invention are variants of the above antibodies, antigen-binding domains and antigen-binding portions.

[0120] Variants of antibodies, antigen-binding domains, or antigen-binding portions thereof described herein have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the non-variant molecule. In one embodiment, the sequence identity is at least 95%. In one embodiment, the modifications (i.e., sequence differences) are conservative sequence modifications.

[0121] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention or antigen-binding portions thereof by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the altered antibodies can be tested for retained function (i.e., CD3 binding) using the functional assays described herein.

[0122] Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired properties of the polypeptide, such as its affinity for an antigen or its specificity. Generally, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids with similar structures or functions are less likely to destroy the biological activity of the polypeptide. Abbreviations for amino acid residues comprising the polypeptides and peptides described herein, and conservative substitutions for these amino acid residues, are provided in Table 3 below.

[0123] Table 3. Examples of amino acid residues and conservative amino acid substitutions TIFF2025540777000001.tif130170

[0124] In some embodiments, the invention provides variants of antibodies, antigen-binding domains or antigen-binding portions thereof, e.g., selected from the sequences shown in Table 3, as compared to the sequences described herein, which comprise one or more sequence modifications and have improved one or more properties, such as binding affinity, specificity, thermal stability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility, as compared to the unmodified antibody or fragment thereof.

[0125] Suitable methods for measuring properties that may indicate that an antigen-binding domain or antibody may be successful in large-scale development include initial purification using chromatography such as affinity chromatography (Protein A: MabSelect Sure LX), anion exchange chromatography (Capto Q), cation exchange chromatography (Capto S), and buffer exchange (G-25 Fine), followed by assessment of antibody integrity (e.g., determination of molecular weight by SDS PAGE analysis, calculation of % monomer by HPLC-SEC, assessment of aggregation, and thermal stability (Tm) testing).

[0126] Those skilled in the art will appreciate that there are various methods (including in vitro and in vivo expression libraries) for identifying, obtaining, and optimizing the antigen-binding molecules described herein. This is further described in the Examples. Optimization techniques known in the art, such as display (e.g., ribosome display and / or phage display) and / or mutagenesis (e.g., error-prone mutagenesis), can be used. Thus, the present invention also encompasses sequence-optimized variants of the antibodies described herein.

[0127] In one embodiment, modifications can be made to reduce the immunogenicity of the antigen-binding domain or antibody. For example, one approach is to revert one or more framework residues to the corresponding canine germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequence of the antibody to the germline sequence from which the antibody is derived. In one embodiment, all framework sequences are germline sequences.

[0128] To return one or more amino acid residues in the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

[0129] Another type of framework modification involves mutating one or more residues within the framework regions, or within one or more CDR regions, to remove T-cell epitopes and thereby reduce the potential immunogenicity of the antigen-binding domain or antibody.

[0130] In some embodiments, antigen-binding proteins, fragments and derivatives thereof, and fusion proteins of the present disclosure undergo post-translational modifications, for example, but not limited to, glutamine being cyclized or converted to pyroglutamic acid. Additionally or alternatively, amino acids may undergo deamidation, isomerization, glycation, and / or oxidation. Polypeptides of the present disclosure may undergo further post-translational modifications, including glycosylation at sites known in the art, e.g., N-linked or O-linked glycosylation. Alterations to the amino acid sequence of the polypeptide can prevent or minimize such alterations or facilitate them in situations where such treatment is beneficial. Polypeptides of the present disclosure include, for example, polypeptides modified to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) impart or modify other physicochemical or functional properties.

[0131] Glycosylation can also be altered to, for example, increase the affinity of the antigen-binding domain or antibody for the antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antigen-binding domain or antibody sequence. For example, one or more amino acid substitutions can be made to remove one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such glycosylation can increase the affinity of the antigen-binding domain or antibody for the antigen.

[0132] In some applications, the antibody, antigen-binding domain, or antigen-binding portion thereof may bind to the desired target (canine CD3 and / or CD20) but has an altered ability to bind to Fc receptors compared to a standard binder. In one example, the binder is an antigen-binding domain or antibody with an altered glycosylation pattern. For example, IgG molecules typically contain N-linked oligosaccharides such as fucose.

[0133] In one embodiment, the antibodies, antigen-binding domains, or antigen-binding portions thereof described herein are α-fucosylated. In cancer immunotherapy, antibodies may rely on antibody-dependent cellular cytotoxicity (ADCC), an Fc-mediated immune effector function, as their primary mechanism of action for depleting tumor cells. This effector function is well known to be regulated by N-linked glycosylation in the Fc region of antibodies. In particular, the absence of core fucose on Fc N-glycans has been shown to increase the binding affinity of IgG1 Fc to FcγRIIIa present on immune effector cells such as natural killer cells, leading to enhanced ADCC activity. Thus, α-fucosylated antigen-binding domains or antibodies may be advantageous for improving therapeutic efficacy, as the absence / removal of fucose enhances the ability of the antigen-binding domain or antibody to interact with Fc receptors. This type of antigen-binding domain or antibody is sometimes referred to as "α-fucosylated." Such antigen-binding domains or antibodies can be produced using techniques described herein and / or known in the art. In some embodiments, nucleic acid sequences encoding antigen-binding domains or antibodies can be expressed in cell lines with altered glycosylation capabilities (e.g., deletions, alterations, or reduced amounts of fucosyltransferase) and incapable of adding typical fucose moieties.

[0134] In one embodiment, the antigen binding domain or the Fc portion of the antibody may be modified.

[0135] In one embodiment, one or more substitutions in the variant are in the CDR1, 2, and / or 3 regions. For example, there may be 1, 2, 3, 4, 5, or more amino acid substitutions in the CDR1, 2, and / or 3 regions. In another example, there may be 1 or 2 amino acid deletions.

[0136] In one embodiment, one or more substitutions are in a framework region, for example, there may be 1 to 20, such as 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in the HC and / or LC variable region framework regions.

[0137] The anti-CD3 antibodies, antigen-binding domains, or portions thereof of the present invention preferably have KD, IC50, and / or EC50 values, such as those further described herein and in the Examples. Suitably, the KD value is sufficient for the antigen-binding domain or antibody to have the desired biological effect. For example, the monovalent KD may be 100 nM to 1000 nM, or less than 100 nM. KD, IC50, and / or EC50 values ​​may be measured as known in the art, for example, as described in the Examples.

[0138] The term "KD" refers to the "equilibrium dissociation constant" and refers to the value obtained by titration measurement at equilibrium or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). "KA" refers to the affinity constant. The association rate constant, dissociation rate constant, and equilibrium dissociation constant are used to express the binding affinity of an antigen-binding domain or antibody to an antigen. Methods for determining the association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to test samples in physiological buffer at equilibrium. Other experimental techniques and instruments, such as the BIAcore® SPR assay, can be used.

[0139] The present invention also relates to isolated canine antibodies, antigen-binding domains, or antigen-binding portions thereof that bind to canine CD3 and compete with the above-described antibodies, antigen-binding domains, or antigen-binding portions thereof. Antibodies, antigen-binding domains, antibody fragments, or antibody mimetics that bind to the same epitope or overlapping epitope on canine CD3 as any of the CD3 antigen-binding domains or antibodies of the present invention have the ability to cross-compete with any of the antigen-binding domains or antibodies of the present invention for binding to CD3. Thus, the antigen-binding domains or antibodies of the present invention can be used as reference antigen-binding domains or antibodies to assess such cross-reactivity. Such cross-competing antigen-binding domains or antibodies can be identified based on their ability to cross-compete with the antigen-binding domains or antibodies described herein in standard CD3 binding assays. For example, BIAcore™ analysis, ELISA assays, or flow cytometry can be used to demonstrate cross-competition with antigen-binding domains or antibodies.

[0140] Immunoconjugates and other binding agents The present invention relates to immunoconjugates and other binding agents comprising an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to CD3 according to the present invention. For example, the antibody, antigen-binding domain, or antigen-binding portion thereof according to the present invention can be conjugated to a therapeutic or non-therapeutic site.

[0141] In one embodiment, the therapeutic moiety is, for example, an antibody, antigen-binding domain, or antibody fragment (e.g., Fab, F(ab'), Fv, single-chain Fv fragment (scFv), or single-domain antibody, e.g., V H or V HH domain), or antibody mimetic proteins, that bind to a target antigen of interest.

[0142] In one embodiment, a protein or polypeptide comprising an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to CD20 as described herein and a second moiety is a fusion protein. In one embodiment, a protein or polypeptide comprising an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to CD3 as described herein and a second moiety is a drug conjugate.

[0143] As used herein, "conjugate" refers to a composition in which an antibody or antigen-binding domain that binds to CD3 as described herein is linked / conjugated to a drug.

[0144] Such conjugates include "drug conjugates" in which the drug comprises a covalently bound antigen-binding domain or antibody that binds to CD3, and "non-covalent drug conjugates" in which the drug comprises a non-covalently bound antigen-binding domain or antibody that binds to CD3.

[0145] As used herein, a "drug conjugate" refers to a composition comprising an antigen-binding domain or antibody to which a drug is covalently bound. The drug can be covalently bound directly or indirectly to the antigen-binding domain, or antibody, or antibody fragment via a suitable linker moiety. The drug can be attached to the antigen-binding domain or antibody at any suitable position, such as the amino terminus, carboxyl terminus, or via a suitable amino acid side chain.

[0146] In one embodiment, the antibody is linked to the second moiety using a peptide linker or other suitable linker to connect the two moieties.

[0147] The term "peptide linker" refers to a peptide comprising one or more amino acids. Peptide linkers comprise 1 to 50, e.g., 1 to 20, amino acids. Peptide linkers are known in the art, and non-limiting examples are provided herein. Suitable non-immunogenic linker peptides include, for example, linkers comprising G and / or S residues, (G4S)n, (SG4)n, or G4(SG4)n peptide linkers, where "n" is generally a number between 1 and 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0148] The binding agent may be multispecific, eg bispecific.

[0149] In one embodiment, the binding molecule is bispecific. Thus, in one aspect, the present invention relates to a bispecific molecule in which an antibody, antigen-binding domain, or antigen-binding portion thereof described herein is linked to a second moiety having a different binding specificity than the antibody, antigen-binding domain, or antigen-binding portion thereof. Thus, the second antibody, antigen-binding domain, or antigen-binding portion thereof binds to a different target antigen, e.g., a target of interest. In one embodiment, the target of interest may be a tumor antigen. In particular, the above-described canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD3 can be used in a bispecific format to target another target, e.g., canine CD20. Such bispecific antigen-binding domains or antibodies are further described below.

[0150] Thus, the present invention also relates to the use of antibodies, antigen-binding domains, or antigen-binding portions thereof that target canine CD3 in bispecific molecules, for example, where the second antigen to be targeted is selected from the following canine antigens: CD20, CD19, CD20, BCMA, CD33, CD38, CEA, CLEC12A, DLL3, EGFRvIII, EpCAM, FcRH5, FLT3, GPC3, gpA33, GPRC5D, HER2, MUC16, P-cadherin, PSMA, SSTR2, CLDN18.

[0151] In one embodiment, the binding molecule, eg, protein or construct, is multispecific and further comprises, eg, third, fourth, fifth, etc. sites.

[0152] The therapeutic moiety may also be selected from a half-life extending moiety, a cytotoxin, or a radioisotope.

[0153] The non-therapeutic moiety can be selected from a label, a liposome, or a nanoparticle. The label is detectable or functional. The label can be any molecule that produces a signal or can be induced to produce a signal, including, but not limited to, a fluorophore, a fluorescent agent, a radioactive label, an enzyme, a chemiluminescent substance, a nuclear magnetic resonance active label, or a photosensitizer. Thus, binding can be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity, or light absorbance.

[0154] According to the present invention, an antibody, antigen-binding domain, or antigen-binding portion linked to one moiety may be further linked to another moiety, for example, may be linked to a therapeutic portion, and further linkage to a non-therapeutic portion may be provided via either the antigen-binding domain, or antibody, or portion.

[0155] In one embodiment, a binding agent according to the invention, or an antibody, antigen-binding domain, or antigen-binding portion thereof, may comprise a half-life extending moiety, which may be selected from an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine serum albumin. Alternatively, the half-life extension may be conferred by PEGylation.

[0156] The term "half-life" as used herein generally refers to the time it takes for the serum concentration of an amino acid sequence, compound, or polypeptide to decrease by 50% in vivo, for example, due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. The in vivo half-life of an amino acid sequence, compound, or polypeptide of the present invention can be determined by any method known per se, such as pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art. Half-life can be expressed using parameters such as t1 / 2-alpha, t1 / 2-beta, and area under the curve (AUC). Half-life (talpha and tbeta) and AUC can be determined from the curve of serum concentration of the conjugate or fusion versus time. Thus, the term "half-life" as used herein particularly refers to t1 / 2-beta or terminal half-life (in which case t1 / 2-alpha and / or AUC, or both, may not be taken into account).

[0157] For example, in the first phase (alpha phase), the drug composition (e.g., drug conjugate, noncovalent drug conjugate, drug fusion) is primarily distributed in the patient's body, with some elimination occurring. The second phase (beta phase) is the terminal phase in which the drug composition (e.g., drug conjugate, noncovalent drug conjugate, drug fusion) is distributed and serum concentration decreases as the drug composition is eliminated from the patient. The talpha half-life is the half-life of the first phase, and the tbeta half-life is the half-life of the second phase.

[0158] Bispecific antibodies targeting canine CD3 and CD20 or antigen-binding portions thereof In another aspect, the present invention relates to a bispecific canine antigen-binding molecule comprising a first antibody, antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD3, in particular CD3εδ, and a second antibody, antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD20. Thus, the bispecific canine antigen-binding molecule comprises one arm (or portion) that specifically binds to canine CD3, and a second arm (or portion) that specifically binds to canine CD20.

[0159] In one embodiment, the bispecific canine antibody, antigen-binding domain, or antigen-binding portion thereof, comprises: a) specifically binds to canine CD3, particularly CD3εδ; b) specifically binds to canine CD20; c) having a CD3-binding arm that binds to canine CD3, particularly CD3εδ, with a monovalent binding dissociation equilibrium constant (KD) of 100 nM to 1000 nM; d) agonistically bind to canine CD3, particularly CD3εδ, activate the canine T cell receptor, and mediate target-specific cell killing in a bispecific format, e.g., as shown in Example 4 in vitro and ex vivo; e) inducing T cell surface upregulation of markers such as CD25 and / or CD69 upon target-mediated cell killing in a bispecific format, as demonstrated, for example, in vitro, ex vivo, and in vivo examples; f) activating canine T cells, e.g., in vitro, with low, e.g., minimal, secretion of cytokines such as IFN-γ, as shown in the Examples, and / or g) have one or more of the following properties: mediating target-specific cell killing in vivo with low / minimal cytokine release, e.g., release of IFN-γ, IL-2, IL-6 and / or TNF-α.

[0160] In one embodiment, the bispecific canine antigen-binding molecule has a monovalent CD3-binding arm that binds to canine CD3, particularly CD3εδ, with a binding dissociation equilibrium constant (KD) of about 100 nM to about 1000 nM.

[0161] Without wishing to be bound by theory, the inventors believe that affinity for canine CD3 within this range provides sufficient binding affinity to elicit agonist activity and mediate cell killing in a bispecific format, while also ensuring low / minimal cytokine release. This is important because cytokine release is one of the primary safety considerations for T cell engager bispecific antibodies. Higher affinity (i.e., lower than about 100 nM) is thought to result in greater cytokine release, which in turn impacts safety.

[0162] In one embodiment, the first antibody, antigen-binding domain, or antigen-binding portion thereof that specifically binds to canine CD3 has a sequence, such as the HCVR / LCVR CDR sequence, HCVR and / or LCVR, described herein and shown in Table 2, which provides the SEQ ID NOs for CDR1, 2, 3, HCVR and LCVR polypeptides.

[0163] In one embodiment, an antigen-binding domain or antigen-binding portion that specifically binds to canine CD3 comprises the HCVR CDRs set forth for a PMX molecule shown in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, a canine antigen-binding domain or antigen-binding fragment portion that specifically binds to canine CD3 comprises or consists of the HCVR CDRs set forth for PMX160, PMX162, PMX169, PMX170, PMX171, PMX172, PMX186, PMX187, PMX190, PMX188, or PMX189, as shown in Table 2.

[0164] In one embodiment, an antigen-binding domain or portion that specifically binds to canine CD3 comprises an HCVR sequence set forth for a PMX molecule shown in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. Thus, in one embodiment, the canine antigen-binding fragment portion comprises an HCVR having an amino acid sequence set forth in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto.

[0165] In one embodiment, the canine antigen-binding domain or antigen-binding fragment portion that specifically binds to canine CD3 comprises or consists of the anti-CD3 HCVR shown for PMX160, PMX162, PMX169, PMX170, PMX171, PMX172, PMX186, PMX187, PMX190, PMX188, or PMX189 shown in Table 2.

[0166] In one embodiment, an antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD3 comprises the LCVR CDRs set forth for a PMX molecule shown in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, a canine antigen-binding domain or antigen-binding fragment portion thereof that specifically binds to canine CD3 comprises or consists of the LCVR CDRs set forth for PMX272, PMX285, PMX286, PMX188, or PMX189, as shown in Table 2.

[0167] In one embodiment, an antigen-binding domain or portion that specifically binds to canine CD3 comprises an LCVR sequence set forth for a PMX molecule shown in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, a canine antigen-binding fragment portion that specifically binds to canine CD3 comprises or consists of the LCVR set forth for PMX272, PMX285, PMX286, PMX188, or PMX189, as shown in Table 2.

[0168] In one embodiment, the second antibody, antigen-binding domain, or antigen-binding portion thereof that specifically binds to canine CD20 has sequences such as the HC / LC CDR sequences, HCVR and / or LCVR described herein and shown in Table 4, which provides the sequence numbers of the CDR1, 2, 3, HCVR and LCVR polypeptides.

[0169] In one embodiment, an antigen-binding domain or antigen-binding portion that specifically binds to canine C20 comprises the HCVR CDRs set forth for a PMX molecule shown in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, the canine antigen-binding fragment portion comprises or consists of the HC CDRs set forth for PMX227-271, as shown in Table 4. In one embodiment, the canine antigen-binding fragment portion comprises VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529.

[0170] In one embodiment, an antigen-binding domain or portion that specifically binds to canine CD20 comprises an HCVR sequence set forth for a PMX molecule shown in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, the canine antigen-binding fragment portion comprises or consists of the HC CDRs set forth for PMX227-271, as shown in Table 4.

[0171] In one embodiment, the canine antigen-binding domain or antigen-binding fragment portion comprises the anti-CD20 HCVR shown for PMX230 shown in Table 4, i.e., SEQ ID NO:524.

[0172] In one embodiment, the antigen-binding portion that specifically binds to canine CD20 comprises an LCVR sequence set forth for a PMX molecule shown in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. Thus, in one embodiment, the canine antigen-binding fragment portion comprises an LCVR having an amino acid sequence shown in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto.

[0173] Thus, according to the invention, any of the anti-CD3 HCVRs of Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto, can be combined in a bispecific molecule with any of the anti-CD20 HCVRs of Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto. Furthermore, any of the anti-CD3 LCVRs of Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto, can be combined in a bispecific molecule with any of the anti-CD20 LCVRs of Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto.

[0174] However, the present inventors have shown that the use of a common light chain can improve the performance of a bispecific molecule. Thus, in one embodiment, the first and second antibodies, antigen-binding domains, or antigen-binding portions thereof, of a bispecific molecule share a common light chain region, e.g., an LCVR or complete light chain. In one embodiment, the LCVR or complete light chain is that of an antibody or antigen-binding domain that binds to canine CD3, e.g., having a SEQ ID NO: described herein, e.g., in Table 2. In one embodiment, the LCVR has an amino acid sequence set forth in Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, the LCVR is that of PMX272, PMX188, PMX189, PMX285, or PMX286, as set forth in Table 2.

[0175] In one embodiment, the bispecific canine antigen-binding molecule comprises a first antibody, antigen-binding domain, or antigen-binding portion thereof, that specifically binds to canine CD3, and a second antibody, antigen-binding domain, or antigen-binding portion thereof, that specifically binds to canine CD20, wherein the antigen-binding molecule is selected from PMX276, PMX277, PMX278, PMX279, PMX280, PMX281, PMX282, PMX283, PMX284, PMX287, or PMX288, as shown in Table 5 below. The components of the CD3- and CD20-binding arms are also shown in this table. Tables 2 and 4 show the SEQ ID NOs corresponding to the CDR1, 2, 3, HCVR, and LCVR polypeptides of each component of the CD3- and CD20-binding arms, with reference to the PMX numbers.

[0176] In one embodiment, the bispecific canine antigen binding molecule(s) comprise: (a) a first antigen binding domain or antigen binding portion thereof that specifically binds to canine CD3 and comprises: (i) a first heavy chain variable region (VH) comprising a VH complementarity Determining Region (CDR)1, a VH CDR2, and a VH CDR3; and (ii) a first light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 68, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 69, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; and (b) a first antigen binding domain or antigen binding portion thereof that specifically binds to canine CD20 and comprises: VH CDR1, VH CDR2, and VH and (ii) a second antigen-binding domain or antigen-binding portion thereof comprising: a second VH comprising a VL CDR1, a VL CDR2, and a VL CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462.

[0177] In another embodiment, the bispecific canine antigen-binding molecule comprises: (a) a first antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD3 and comprises: (i) a first heavy chain variable region (VH) comprising a VH complementarity-Determining Region (CDR)1, a VH CDR2, and a VH CDR3; and (ii) a first light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 87, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 88, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 89, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; and (b) a first antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD20 and comprises: VH CDR1, VH CDR2, and VH and (ii) a second antigen-binding domain or antigen-binding portion thereof comprising: a second VH comprising a VL CDR1, a VL CDR2, and a VL CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462.

[0178] In yet another embodiment, the bispecific canine antigen-binding molecule comprises: (a) a first antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD3 and comprises: (i) a first heavy chain variable region (VH) comprising a VH complementarity-Determining Region (CDR)1, a VH CDR2, and a VH CDR3; and (ii) a first light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 157, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 158, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 159, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; and (b) a first antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD20 and comprises: VH CDR1, VH CDR2, and VH and (ii) a second antigen-binding domain or antigen-binding portion thereof comprising: a second VH comprising a VL CDR1, a VL CDR2, and a VL CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462.

[0179] In one embodiment, the bispecific canine antigen-binding molecule comprises: (a) a first antigen-binding domain, or antigen-binding portion thereof, that specifically binds to canine CD3 and comprises: (i) a first VH comprising VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 167, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 168, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 169, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; and (b) a second VH that specifically binds to canine CD20 and comprises: (i) a VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3. and a second VL comprising VH CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462.

[0180] In another embodiment, the bispecific canine antigen-binding molecule comprises: (a) a first antigen-binding domain, or antigen-binding portion thereof, that specifically binds to canine CD3 and comprises: (i) a first VH comprising VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 177, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 178, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 179, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; and (b) a second VH that specifically binds to CD20 and comprises: (i) a VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3. and a second antigen-binding domain or antigen-binding portion thereof comprising a second VL comprising VH CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462.

[0181] In yet another embodiment, the bispecific canine antigen-binding molecule comprises: (a) a first antigen-binding domain, or antigen-binding portion thereof, that specifically binds to canine CD3 and comprises: (i) a first VH comprising VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 187, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 188, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 189, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; and (b) a second VH that specifically binds to CD20 and comprises: (i) a VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3. and a second antigen-binding domain or antigen-binding portion thereof comprising a second VL comprising VH CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462.

[0182] In one embodiment, the bispecific canine antigen binding molecule comprises: (a) a first antigen binding domain, or antigen binding portion thereof, that specifically binds to canine CD3 and comprises: (i) a first VH comprising VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 327, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 328, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 329, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 480, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 481, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 482; and (b) a second VH that specifically binds to CD20 and comprises: (i) a VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3. and a second antigen-binding domain or antigen-binding portion thereof comprising a second VL comprising VH CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 480, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 481, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 482.

[0183] In another embodiment, the bispecific canine antigen-binding molecule comprises: (a) a first antigen-binding domain, or antigen-binding portion thereof, that specifically binds to canine CD3 and comprises: (i) a first VH comprising VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 337, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 338, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 339, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 490, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 491, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 492; and (b) a second VH that specifically binds to CD20 and comprises: (i) a VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3. and a second antigen-binding domain or antigen-binding portion thereof comprising a second VL comprising VH CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 490, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 491, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 492.

[0184] In yet another embodiment, the bispecific canine antigen-binding molecule comprises: (a) a first antigen-binding domain, or antigen-binding portion thereof, that specifically binds to canine CD3 and comprises: (i) a first VH comprising VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 357, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 358, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 359, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; and (b) a second VH that specifically binds to CD20 and comprises: (i) a VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3. and a second antigen-binding domain or antigen-binding portion thereof comprising a second VL comprising VH CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462.

[0185] In one embodiment, the bispecific canine antigen binding molecule comprises: (a) a first antigen binding domain, or antigen binding portion thereof, that specifically binds to canine CD3 and comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 347, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 348, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 349, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 350, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 351, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 352; and (b) a second VH that specifically binds to CD20 and comprises: (i) a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3. and a second antigen-binding domain or antigen-binding portion thereof comprising a second VL comprising VH CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 350, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 351, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 352.

[0186] In another embodiment, the bispecific canine antigen-binding molecule comprises: (a) a first antigen-binding domain, or antigen-binding portion thereof, that specifically binds to canine CD3 and comprises: (i) a first VH comprising VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 107, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 108, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 109, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 110, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 111, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 112; and (b) a second VH that specifically binds to CD20 and comprises: (i) a VH CDR1, VH CDR2, and VH CDR3; and (ii) a first VL comprising VL CDR1, VL CDR2, and VL CDR3. and a second antigen-binding domain or antigen-binding portion thereof comprising a second VL comprising VH CDR1, VL CDR2, and VL CDR3, wherein VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, VL CDR1 comprises the amino acid sequence of SEQ ID NO: 110, VL CDR2 comprises the amino acid sequence of SEQ ID NO: 111, and VL CDR3 comprises the amino acid sequence of SEQ ID NO: 112.

[0187] In one embodiment, the bispecific canine antigen-binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 64, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 456. In another embodiment, the bispecific canine antigen-binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 84, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 456. In yet another embodiment, the bispecific canine antigen-binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 154, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 456. In one embodiment, the bispecific canine antigen-binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 164, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 456. In one embodiment, the bispecific canine antigen-binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 174, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 456. In another embodiment, the bispecific canine antigen-binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 184, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 456.In yet another embodiment, the bispecific canine antigen binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 324, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 476. In one embodiment, the bispecific canine antigen binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 334, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 486. In another embodiment, the bispecific canine antigen-binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 74, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 456. In yet another embodiment, the bispecific canine antigen-binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 344, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 346. In one embodiment, the bispecific canine antigen-binding molecule comprises a) a first VH that specifically binds to canine CD3 comprising the amino acid sequence of SEQ ID NO: 104, b) a second VH that specifically binds to CD20 comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL that bind to CD3 and / or CD20 comprising the amino acid sequence of SEQ ID NO: 106.

[0188] Thus, in one embodiment, the bispecific molecule comprises: a) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX160 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX272 in Table 2; b) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX162 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX272 in Table 2; c) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX169 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX272 in Table 2; d) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX170 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX272 in Table 2; e) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX171 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX272 in Table 2; f) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX172 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX272 in Table 2; g) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX186 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX285 in Table 2; h) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX187 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX286 in Table 2; i) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX190 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX272 in Table 2; j) a canine CD3-binding HCVR having the SEQ ID NO: shown for PMX188 in Table 2, a canine CD20-binding HCVR having the SEQ ID NO: shown for PMX230 in Table 4, and a consensus LCVR shown for PMX188 in Table 2; or k) Comprises an HCVR that binds to canine CD3 having the sequence number shown for PMX189 in Table 2, an HCVR that binds to canine CD20 having the sequence number shown for PMX230 in Table 4, and a consensus LCVR shown for PMX189 in Table 2.

[0189] Table 5. Exemplary bispecific molecules TIFF2025540777000002.tif78155

[0190] Bispecific antibodies or antigen-binding molecules according to the invention are not limited to any particular bispecific format or method of production thereof.

[0191] Examples of bispecific antibodies or antigen-binding molecules that can be used in the present invention include (i) a single antibody or antigen-binding domain with two arms containing different antigen-binding regions, (ii) a single antibody or antigen-binding domain with specificity for two different epitopes, for example, via two scFvs linked in tandem by an additional peptide linker, (iii) dual variable domain antibodies (DVD-Ig) in which each light and heavy chain contains two variable domains in tandem via a short peptide bond, (iv) chemically linked bispecific (Fab')2 fragments, and (v) tetraspecific antibodies with two binding sites for each target antigen. (vi) flexibodies, which combine scFvs and diabodies to form multivalent molecules; (vii) so-called "dock-and-lock" molecules, based on the "dimerization and docking domain" of protein kinase A, which when applied to Fabs can generate trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called scorpion molecules, which contain, for example, two scFvs fused to either end of a human Fab arm; and (ix) diabodies.

[0192] In one embodiment, the antibody, antigen-binding domain, or antigen-binding portion thereof comprises an Fc region, such as a canine Fc region, such as a canine IgGB Fc region. In one embodiment, the bispecific antibody or antigen-binding molecule of the invention comprises a first Fc region comprising a first CH3 region and a second Fc region comprising a second CH3 region. In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises first and second heavy chains, each of said first and second heavy chains comprising at least a hinge region, a CH2 region, and a CH3 region.

[0193] Bispecific antibodies or antigen-binding molecules according to the present invention may contain modifications in the Fc region. Thus, in the context of bispecific antigen-binding molecules of the present invention, the multimerization domain, e.g., the Fc domain, may contain one or more amino acid changes (e.g., insertions, deletions, or substitutions) compared to a wild-type, naturally occurring version of the Fc domain. For example, the present invention includes bispecific antigen-binding molecules containing one or more modifications in the Fc domain, resulting in a modified Fc domain with altered (e.g., enhanced or decreased) binding interactions between the Fc and Fc receptors. In one embodiment, the bispecific antigen-binding molecule contains a modification in the CH2 or CH3 region.

[0194] The variable region sequences described herein, including but not limited to the amino acid and nucleotide sequences (and / or fragments thereof) set forth in Tables 2 and 4, can be used in combination with one or more amino acid and / or nucleotide sequences encoding one or more constant chains (and / or fragments thereof) of an antibody molecule. For example, the variable region amino acid sequences set forth in Tables 2 or 4 can be linked to any antigen-binding domain or constant region of an antibody molecule from the same or a different species (e.g., human, goat, rat, sheep, chicken) as the species from which the variable region amino acid sequence is derived. Preferably, the variable region amino acid sequences set forth in Tables 2 or 4 are linked to a canine antigen-binding domain or constant region of a canine antibody, which may be the constant region of any of canine IgG A, B, C, or D. In one embodiment, the constant region is a canine IgG B constant region. The constant regions of canine IGGB (SEQ ID NO: 28), canine IGK, or canine IGLC5 may be used. Variants of constant regions with modified effector regions, such as a variant of canine IGGB (SEQ ID NO: 30), may also be used. These variants can be formed by introducing mutations that neutralize effector function into canine IgG-B. The canine IgG-B can be modified to reduce or neutralize the effector function of the canine IgG-B when compared to the same polypeptide containing a wild-type IgG-B Fc domain. Regions of the amino acid sequence of the Fc domain that can be modified include the lower hinge, proline region, and SHED region, where interactions with FcgammaR and C1q may occur. Examples of such mutations are described in WO2023 / 012486, which is incorporated herein by reference.

[0195] Other such variants may include charge pair combinations in the canine CH3 domain, which may significantly enhance heavy chain heterodimerization over homodimer formation. This can minimize the formation of homodimer contaminants in the production of bispecific antibodies. These charge pair combinations may be present within the canine IgG CH3 domain interface of the Fc region, where the IgG is selected from IgG-A, B, C, or D. Both the first canine IgG CH3 domain and the second canine IgG CH3 domain may be complementarily designed so that each CH3 domain (or polypeptide containing it) does not substantially homodimerize with itself or homodimerizes at a slower rate, but is forced to heterodimerize with the other complementarily designed CH3 domain. In other words, the first and second CH3 domains may form heterodimers, and homodimers between two first or two second CH3 domains are rarely formed. Examples of such mutations are described in WO2021 / 214460 A1, incorporated herein by reference.

[0196] Variants may also contain mutations in the canine CH2 or CH3 IgG Fc domain that result in differential affinity for binding affinity reagents and / or improved stability. For example, a binding molecule may have differential affinity for binding to Protein A compared to a wild-type IgG Fc domain. Differential affinities of immunoglobulin heavy chains allow for optimal isolation of the binding protein or antibody. For example, a suitable variant IgG Fc domain may contain one or more amino acid substitutions that increase the affinity for binding to Protein A or one or more amino acid substitutions that decrease the affinity for binding to Protein A. Examples of such mutations are described in GB2311984.5, which is incorporated herein by reference.

[0197] Thus, in one embodiment, the antibody, antigen-binding domain, or antigen-binding portion thereof, comprises an Fc-binding-deficient variant resulting from mutation of canine IgG-B. In another embodiment, the antibody, antigen-binding domain, or antigen-binding portion thereof, comprises an enhanced heavy chain heterodimerization variant resulting from mutation of the canine CH3 domain of canine IgG-A, B, C, or D. In another embodiment, the antibody, antigen-binding domain, or antigen-binding portion thereof, comprises a variant with mutations in the canine CH2 or CH3 IgG Fc domain that result in differential affinity for binding affinity reagents and / or improved stability. The antibody, antigen-binding domain, or antigen-binding portion thereof may have a single mutation that results in a single mutant phenotype (e.g., reduced Fc effector function) or may have multiple mutations that result in multiple phenotypes (e.g., reduced Fc effector function, enhanced heavy chain heterodimerization, and altered Protein A binding affinity).

[0198] Canine antibodies that bind to canine CD20 or antigen-binding portions thereof As mentioned above, the present invention relates to canine antibodies, antigen-binding domains, or antigen-binding portions thereof that bind to canine CD3 and canine CD20. Table 4 provides examples of canine antibodies, antigen-binding domains, or antigen-binding portions thereof that bind to canine CD20 and can be used in such bispecific molecules.

[0199] In yet another aspect, the present invention also relates to canine antibodies, antigen-binding domains, or antigen-binding portions thereof that bind to canine CD20. Such antibodies, antigen-binding domains, or antigen-binding portions thereof can be used in monovalent or bispecific formats, e.g., with the CD3 antibodies described above.

[0200] In one embodiment, an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD20 according to the present invention is a) specifically binds to canine CD20; b) binds to canine CD20 with a KD as measured in the examples, e.g., as shown in the figures; c) exhibiting cell killing, such as CDC and / or ADCC, in a CD20-expressing canine lymphoma cell line, as measured in the Examples; d) promoting antibody-dependent cellular phagocytosis (ADCP); e) It can effectively deplete CD20-positive B cells in dog tissues; f) capable of binding to cells expressing canine CD20, and / or g) capable of depleting cells expressing canine CD20, suitably by direct cell killing via apoptosis.

[0201] In one embodiment, an antibody, antigen-binding domain, or antigen-binding portion thereof according to the invention has one or more of the above properties, and optionally a) have CDC activity with an EC50 value of less than 20 nM, and / or b) having ADCC activity with an EC50 value of less than 0.3 nM;

[0202] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment portion that binds to canine CD20 is selected from the group consisting of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX260, PMX261, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, PMX269, PMX270, PMX271, PMX272, PMX273, PMX274, PMX275, PMX276, PMX277, PMX278, PMX279, PMX280, PMX281, PMX282, PMX283, PMX284, PMX285, PMX286, PMX287, PMX288, PMX289, PMX290, PMX291, PMX292, PMX293, PMX294, PMX295, PMX296, PMX297, PMX298, PMX299, PMX300, PMX301, PMX302, PMX303, PMX304, PMX305, PMX306, PMX307, PMX308, PMX309, PMX310, PMX311, PMX312, PMX313, PMX314, PMX315, PMX316, P The antibody comprises a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 4 for PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. Complementarity-determining region (CDR) refers to the three CDRs, i.e., CDR1, 2, and 3.

[0203] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment thereof is selected from the group consisting of (a) PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX2 a heavy chain variable region having an amino acid sequence set forth in Table 4 for PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto; (b) the complementarity-determining regions (CDRs) of the HCVR region, and (c) PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257 7, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or the CDRs of a light chain variable region (LCVR) having an amino acid sequence set forth in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto.

[0204] In other words, in one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment portion thereof comprises (a) a HC1 antibody or HC2 antibody fragment thereof as shown for one of the PMX molecules in Table 4 for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269. and (b) a CDR or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto, and (c) a LC of the light chain variable region (LCVR) as shown for one of the PMX molecules in Table 4 for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269. It comprises a CDR or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto. In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment thereof comprises the HC CDRs and LC CDRs of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, as shown in Table 4.

[0205] In one embodiment, the present invention relates to an isolated canine antibody, antigen-binding domain, or antigen-binding portion thereof, that binds to canine CD20, said antibody, antigen-binding domain, or antigen-binding portion thereof comprising: a) a heavy chain (HC) CDR1 sequence comprising or consisting of a SEQ ID NO: set out for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 in Table 4, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto; b) an HC CDR2 sequence comprising or consisting of the SEQ ID NO set forth for each of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 in Table 4, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto; c) an HC CDR3 sequence comprising or consisting of the SEQ ID NO set forth for each of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 in Table 4, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto; d) a light chain (LC) CDR1 sequence comprising or consisting of SEQ ID NO: PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 in Table 4, or an amino acid sequence having at least 60%, 70%, 80%, or 90% sequence identity thereto; e) an LC CDR2 sequence comprising or consisting of SEQ ID NO: PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 in Table 4, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto; and f) An LC CDR3 sequence comprising or consisting of SEQ ID NO: PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 in Table 4, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto.

[0206] In one embodiment, an isolated canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD20, wherein said antibody, antigen-binding domain, or antigen-binding portion thereof is a) a HC CDR1 sequence comprising or consisting of a SEQ ID NO: set out for a PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 molecule in Table 4; b) a HC CDR2 sequence comprising or consisting of the SEQ ID NO: shown for each PMX molecule in Table 4; c) a HC CDR3 sequence comprising or consisting of the SEQ ID NO: shown for each PMX molecule in Table 4; d) an LC CDR1 sequence comprising or consisting of the SEQ ID NO: set out for each PMX molecule in Table 4; e) an LC CDR2 sequence comprising or consisting of the SEQ ID NO: set out for each PMX molecule in Table 4, and f) comprises an LC CDR3 sequence comprising or consisting of a SEQ ID NO: set out for each PMX molecule in Table 4, Alternatively, it is an isolated canine antibody or antigen-binding portion thereof having the above CDRs, but having one or more CDRs with 1, 2, 3, 4, or 5 amino acid substitutions in one or more LC or HC CDRs compared to a reference CDR sequence.

[0207] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment portion is selected from the group consisting of (a) PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX260, PMX261, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, PMX269, PMX270, PMX271, PMX272, PMX273, PMX274, PMX275, PMX276, PMX277, PMX278, PMX279, PMX280, PMX281, PMX282, PMX283, PMX284, PMX285, PMX286, PMX287, PMX288, PMX289, PMX290, PMX291, PMX292, PMX293, PMX294, PMX295, PMX296, PMX297, PMX298, PMX299, PMX300, PMX301, PMX302, PMX303, PMX304, PMX305, PMX306, PMX307, PMX308, PMX309, PMX310, PMX311, PMX312, PMX313, PMX314, PMX315, PMX316, PMX317, P 255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. and (b) a heavy chain variable region (HCVR) corresponding to PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, and a light chain variable region (LCVR) having an amino acid sequence set forth for PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment thereof comprises the HC CDRs and LC CDRs of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, as shown in Table 4.

[0208] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding fragment thereof comprises the HCVRs and LCVRs of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, as shown in Table 4.

[0209] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding portion thereof is a) an HCVR sequence comprising or consisting of a SEQ ID NO: PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 in Table 4, or a sequence which has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations (e.g., deletions, additions, or substitutions) in the HCVR framework regions compared to a reference HCVR; b) an LCVR sequence comprising or consisting of a sequence number set forth for the respective PMX molecule in Table 4, or a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications (e.g., deletions, additions or substitutions) in the LCVR framework regions compared to a reference LCVR.

[0210] In one embodiment, the antigen-binding portion is a F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy chain (V H ) domain, or the variable light chain (V L )

[0211] In one embodiment, the antigen-binding domain or portion is a heavy chain and comprises the HC CDRs set forth for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 shown in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereof. In one embodiment, the canine antigen-binding domain or antigen-binding fragment portion comprises or consists of the HC CDRs set out for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 in Table 4.

[0212] In one embodiment, the antigen binding domain or portion is a heavy chain variable region and comprises an HCVR as set forth for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 as shown in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto. Thus, in one embodiment, the canine antigen-binding domain or antigen-binding fragment portion comprises an HCVR having the amino acid sequence of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 shown in Table 4, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity thereto.

[0213] In one embodiment, the canine antigen-binding domain or antigen-binding fragment portion comprises or consists of the HCVRs set forth for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 in Table 4.

[0214] In another embodiment, the invention relates to the use of these antibodies, antigen-binding domains or antigen-binding portions thereof in bispecific antibodies together with antibodies, antigen-binding domains or antigen-binding portions thereof that bind to CD20.

[0215] In one embodiment, the above-described antibody, or antigen-binding portion thereof, comprises an Fc region, eg, a canine Fc region, eg, a canine IgGB Fc region.

[0216] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD20 comprises the VH sequence shown for PMX230 or a VH with greater than 80% identity.

[0217] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding portion thereof is an scFv, Fv, heavy chain, or single domain antibody.

[0218] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding portion thereof is conjugated to a therapeutic moiety.

[0219] In one embodiment, the therapeutic moiety is a second or further antibody, antigen-binding domain, or antigen-binding portion thereof.

[0220] In one embodiment, the second antibody, antigen-binding domain, or antigen-binding portion thereof binds to a different target, e.g., a target that is not CD3 or CD20. In one embodiment, the different target is a tumor antigen.

[0221] In one embodiment, the canine antibody, antigen-binding domain, or antigen-binding portion thereof is conjugated to an additional moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope.

[0222] The present invention relates to immunoconjugates and other binding agents comprising an antibody, antigen-binding domain, or antigen-binding portion thereof according to the invention that binds to CD20. For example, the antibody, antigen-binding domain, or antigen-binding portion thereof according to the invention may be conjugated to a therapeutic or non-therapeutic moiety.

[0223] Pharmaceutical Composition In another aspect, there is provided a pharmaceutical composition comprising an antibody, antigen-binding domain, or fragment of the present invention, i.e., a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD3, a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to both canine CD3 and CD20, or a canine antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD20, and optionally a pharmaceutically acceptable carrier. As used herein, the term pharmaceutical composition refers to a composition used to treat companion animals and for veterinary use, i.e., a veterinary composition. In a preferred embodiment, the animal to be treated is a dog.

[0224] The pharmaceutical composition may optionally comprise a pharmaceutically acceptable carrier. The antibody, protein or construct, or pharmaceutical composition may be administered by any convenient route, including, but not limited to, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topically (particularly to the ear, nose, eye, or skin, or by inhalation).

[0225] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, or subcutaneous administration. Preferably, the compositions are administered parenterally.

[0226] The pharmaceutically acceptable carrier or vehicle may be particulate, so that the composition is, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant, or excipient with which the drug-antibody conjugate of the present invention is administered. Such pharmaceutical carriers may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Carriers may also be saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. Additionally, auxiliary substances, stabilizers, thickeners, lubricants, and coloring agents may be used. In one embodiment, the antigen-binding domain or antibody of the present invention, or composition and pharmaceutically acceptable carrier are sterile when administered to an animal. Water is a preferred carrier when the drug-antibody conjugate of the present invention is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0227] The pharmaceutical compositions of the present invention may be in the form of a liquid, such as a solution, emulsion, or suspension. The liquid may be useful for injection, infusion (e.g., intravenous infusion), or subcutaneous administration. When intended for oral administration, the composition is preferably in solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included within the forms considered herein as either solid or liquid.

[0228] As a solid composition for oral administration, the composition can be formulated in the form of powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose, or dextrin; disintegrants such as alginic acid, sodium alginate, or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavors such as peppermint, methyl salicylate, or orange flavor; and colorants. When the composition is in the form of a capsule (e.g., a gelatin capsule), in addition to the above-mentioned materials, it can contain a liquid carrier such as polyethylene glycol, cyclodextrin, or fatty oil.

[0229] The composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. This liquid may be useful for oral administration or delivery by injection. When intended for oral administration, the composition may contain one or more of a sweetener, a preservative, a dye / colorant, and a flavor enhancer. In a composition for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent may also be included.

[0230] The compositions may be in the form of one or more dosage units. In certain embodiments, it may be desirable to administer the compositions locally to the area in need of treatment or by intravenous injection or infusion.

[0231] How to Treat a Disease The present invention further extends to methods of treating disease, the administration of a pharmaceutical composition or formulation described herein, or an antibody, antigen-binding domain, or antigen-binding portion thereof of the invention, i.e., a canine antibody that binds to canine CD3, an antigen-binding domain, or antigen-binding portion thereof, a canine antibody that binds to both canine CD3 and CD20, an antigen-binding domain, or antigen-binding portion thereof, or a canine antibody that binds to canine CD20, an antigen-binding domain, or antigen-binding portion thereof. Also contemplated are binding molecules or fusion proteins comprising a pharmaceutical composition or formulation described herein, or an antibody, antigen-binding domain, or antigen-binding portion thereof, described herein, i.e., a canine antibody that binds to canine CD3, an antigen-binding domain, or antigen-binding portion thereof, a canine antibody that binds to both canine CD3 and CD20, an antigen-binding domain, or antigen-binding portion thereof, or a canine antibody that binds to canine CD20, for use in treating disease.

[0232] In particular, canine antibodies, antigen-binding domains, or antigen-binding portions thereof that bind to canine CD3 can be used to treat canine diseases such as autoimmune diseases, e.g., type 1 diabetes and graft-versus-host disease. In particular, bispecific antibodies that bind to both canine CD3 and CD20, or canine antibodies, antigen-binding domains, or antigen-binding portions thereof that bind to canine CD20, can be used to treat B-cell mediated conditions.

[0233] In particular, the present invention relates to a method of treating a B-cell mediated condition in a canine subject in need thereof, comprising administering an effective amount of an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to CD20 as described herein.

[0234] An aspect of the invention is also an antibody, antigen-binding domain or antigen-binding portion that binds CD20, or a pharmaceutical composition as described herein, for use in treating a B-cell mediated condition in a canine subject.

[0235] For example, the antibodies, antigen-binding domains, or antigen-binding portions thereof can be used to deplete B-cell lymphoma cells from the blood and / or tissues of a dog. The B-cell mediated condition is selected from B-cell lymphoma (e.g., diffuse large B-cell lymphoma, Hodgkin's lymphoma and non-Hodgkin's lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), small cell lymphocytic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis), leukemia, or an immune-mediated disease. The immune-mediated disease may be an autoimmune disease. Examples include, but are not limited to, autoimmune hemolytic anemia, immune-mediated thrombocytopenia, autoimmune bullous diseases, immune-mediated arthritis and atopic dermatitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjogren's syndrome, vasculitis, multiple sclerosis, Graves' disease, idiopathic thrombocytopenia, dermatomyositis, immune-mediated thrombocytopenia, polymyocytosis, pemphigus, immune-mediated hemolytic anemia, and bullous pemphigoid.

[0236] The amount of therapeutic agent that is effective / active in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be used to help identify optimal dosage ranges. The exact dose to be employed in the composition will also depend on the route of administration and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors such as age, weight, sex, diet, time of administration, excretion rate, condition of the host, drug combinations, reaction sensitivities, and severity of the disease must be taken into consideration.

[0237] Typically, the amount is at least about 0.01% of the antibody, antigen-binding domain, or fragment thereof of the present invention by weight of the composition. When intended for oral administration, this amount can vary from about 0.1% to about 80% by weight of the composition. Preferred oral compositions may contain about 4% to about 50% of the antibody or fragment thereof of the present invention by weight of the composition.

[0238] A preferred composition of the present invention is prepared so that a parenteral dosage unit contains about 0.01% to about 2% by mass of the antibody, antigen-binding domain, or fragment thereof of the present invention.

[0239] For administration by injection, such as intravenous or subcutaneous injection, the composition typically comprises about 0.01 mg / kg to about 250 mg / kg of the subject's body weight, e.g., 0.1 mg / kg to about 250 mg / kg, e.g., about 0.1 mg / kg to about 20 mg / kg of the animal's body weight, more preferably about 1 mg / kg to about 10 mg / kg of the animal's body weight, although amounts less than 0.1 mg / kg are contemplated. In one embodiment, the composition is administered at a dose of about 0.5 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 0.5 to 5 mg / kg, about 0.5 to 2.5 mg / kg, about 0.5 to 2.0 mg / kg, or about 2 or 3 mg / kg. In one embodiment, the composition is administered at a dose of 2 to 50 mg / ml. In one embodiment, the composition is administered at a dose of 0.5 mg / ml to 2.5 mg / ml, or 0.5 mg / ml to 5 mg / ml. The dosing schedule can vary, for example, from once a week to once every 2, 3, or 4 weeks, or up to 8 weeks between doses. In one embodiment, the composition is administered at a dose of 0.5 mg / ml to 2.5 mg / ml every 3 to 4 weeks, for example, 0.5 mg / ml or 2.5 mg / ml every 3 to 4 weeks. Suitably, the dose is selected to provide long-term depletion of CD20-positive cells, with a 3 to 4 week interval between doses. Multiple doses can be administered, suitably up to about 6 or more repeated doses.

[0240] In one embodiment, after treatment, the subject is free of disease progression for at least 7 days, or at least 14 days, or at least 21 days, or at least 28 days, or at least 40 days, or at least 50 days, or at least 60 days. In one embodiment, after treatment, the subject is free of disease progression for at least 7 days, or at least 14 days, or at least 21 days, or at least 28 days, or at least 40 days, or at least 50 days, or at least 60 days.

[0241] In one embodiment, the number of days alive, disease free or disease progression free is at least 2 months, or at least 3 months, or at least 4 months, such as at least 5 months, for example at least 6 months.

[0242] In one embodiment, the number of days alive, disease-free, or disease progression-free is at least 9 months, 200 days, 300 days, or 3 years or more. In one embodiment, it is at least 1 year, 2 years, 3 years, or more. The invention provides a method of treating or preventing a CD3- and / or CD20-mediated disease or disorder in a companion animal, e.g., a dog, comprising administering to the animal in need thereof an effective amount of an antibody, antigen-binding domain, or fragment of the invention.

[0243] As used herein, "treat," "treating," or "treatment" means inhibiting or alleviating a disease or disorder. For example, treatment can include delaying the onset of symptoms associated with a disease or disorder and / or reducing the severity of such symptoms that manifest or are expected to manifest with the disease. These terms include amelioration of existing symptoms, prevention of additional symptoms, and amelioration or prevention of the underlying causes of such symptoms. Thus, these terms indicate that a beneficial result has been achieved in at least some of the treated mammalian, e.g., canine, patients. Many medical treatments are effective in some, but not all, patients receiving the treatment. For example, in the treatment of B-cell lymphoma, improvement in symptoms can be assessed by measuring lymph nodes after treatment and observing a decrease in lymph node size as an indicator of successful treatment.

[0244] The term "subject" or "patient" refers to a dog that is the object of treatment, observation, or experiment. For the avoidance of doubt, the treatment of humans is excluded.

[0245] The molecules or pharmaceutical compositions of the present invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agents, such as cancer therapy. In some embodiments, the cancer therapy is radiation therapy. The therapeutic agent is a compound or molecule useful in treating a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, proapoptotic agents, antiangiogenic agents, boron compounds, photoactive agents or dyes, radioisotopes, immunosuppressants, or immunomodulators such as cytokines or chemokines. In one example, the molecules or pharmaceutical compositions of the present invention may be administered more or less weekly over a period of several months in combination with a multi-drug CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisone)-based chemotherapy protocol incorporating several injectable and oral agents (lasparaginase, vincristine, cytoxan, prednisone, and doxorubicin). Administration can be simultaneous with, before, or after administration of the compounds of the present invention.

[0246] Accordingly, the present invention also relates to combination therapies comprising an antibody, antigen-binding domain, or antigen-binding portion thereof, that binds to CD3 described herein, a bispecific antibody described herein, or a pharmaceutical composition described herein, and an additional therapeutic moiety. The additional therapeutic moiety can be an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD20. Accordingly, the present invention relates to combination therapies comprising a bispecific antibody described herein and an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to canine CD20. Accordingly, the present invention also relates to combination therapies comprising an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to CD3 described herein, and, for example, an antibody, antigen-binding domain, or antigen-binding portion thereof that binds to CD20 described herein. The antibody, antigen-binding domain, or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition, and the additional therapeutic moiety are administered simultaneously or sequentially.

[0247] The present invention also relates to a method of inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of an antibody, antigen-binding domain or antigen-binding portion thereof, or pharmaceutical composition described herein. The method can be in vitro, in vivo, or ex vivo.

[0248] The present invention also relates to a method for killing tumor cells that express CD20, comprising contacting the cells with an antibody or pharmaceutical composition described herein, thereby killing the cells that express CD20. The tumor cells are canine tumor cells. The method can be in vitro, in vivo, or ex vivo.

[0249] Also provided are methods for eliminating cells expressing canine CD20 using the antibodies, antigen-binding domains, or pharmaceutical compositions described herein, which may be in vitro, in vivo, or ex vivo.

[0250] Nucleic acid sequences, vectors and host cells The present invention also relates to nucleic acid sequences encoding the amino acid sequences of canine antibodies or antigen-binding portions thereof that bind to CD3 described herein, e.g., the HC variable region or the LC variable region. Exemplary sequences are set forth in Table 2. In one embodiment, the nucleic acid is selected from a sequence set forth in Table 2, or a nucleic acid having at least 75%, 80%, or 90% sequence identity thereto.

[0251] The invention also relates to nucleic acid sequences encoding the amino acid sequence of the HC variable region or LC variable region of a canine antibody, or antigen-binding portion thereof, that binds to CD20 as described herein, e.g., PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269 shown in Table 4.

[0252] The present invention also relates to nucleic acid sequences encoding the amino acid sequence of a canine bispecific antibody or antigen-binding portion thereof that binds to CD3 and CD20 as described herein, e.g., the HC variable region or the LC variable region. Exemplary sequences are set forth in Table 2. In one embodiment, the nucleic acid encoding the CD3-binding portion is selected from the sequences set forth in Table 2, or a nucleic acid having at least 75%, 80%, or 90% sequence identity thereto. In one embodiment, the nucleic acid encoding the CD20-binding portion is selected from the sequences set forth in Table 4, or a nucleic acid having at least 75%, 80%, or 90% sequence identity thereto.

[0253] In one embodiment, the nucleic acid sequence is linked to a second nucleic acid sequence via a linker. In one embodiment, the second nucleic acid encodes an additional therapeutic moiety. In one embodiment, the linker is a nucleic acid linker. Exemplary nucleic acids are shown below. However, one skilled in the art will understand that due to the degeneracy of the genetic code, other sequences are contemplated and within the scope of the present invention.

[0254] Nucleic acids according to the invention may comprise DNA or RNA and may be wholly or partially synthetically or recombinantly produced. Reference to a nucleotide sequence herein encompasses DNA molecules having the particular sequence and, unless the context indicates otherwise, also encompasses RNA molecules having the particular sequence in which U is replaced by T.

[0255] Furthermore, the present invention relates to a nucleic acid construct comprising at least one nucleic acid as defined above, said construct being in the form of a plasmid, a vector, a transcription cassette or an expression cassette.

[0256] The present invention also relates to vectors containing nucleic acids encoding the CD3 or CD20 antibodies, antigen-binding domains, or antigen-binding portions thereof described herein. The term "vector" refers to a nucleic acid molecule, preferably a DNA molecule derived from, for example, a plasmid, bacteriophage, or virus, into which a nucleic acid sequence can be inserted or cloned. Vectors preferably contain one or more unique restriction enzyme sites and are capable of autonomous replication in a defined host cell, including a target cell or tissue, or its progenitor cell or tissue, or can integrate into the genome of a defined host so that cloned sequences can be reproduced. Thus, a vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a linear or closed circular plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. A vector may contain any means for ensuring self-replication. Alternatively, a vector may be integrated into the genome of a host cell upon introduction and replicated along with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The selection of vector usually depends on the compatibility of the vector with the host cell into which the vector is introduced.The vector can also contain a selection marker, such as an antibiotic resistance gene, which can be used to select suitable transformants.Examples of such resistance genes are well known to those skilled in the art.In one embodiment, the vector is an adeno-associated virus (AAV) vector, such as that described in WO2021176362.

[0257] In some embodiments, the nucleic acid may also include a leader sequence. In other embodiments, the nucleic acid does not include a leader sequence. To enhance protein expression, appropriate leader sequences may be selected, including native immunoglobulin germline leader sequences such as SEQ ID NO: 943 (MESALSWVFLVTILKGVQG) for the heavy chain and SEQ ID NO: 944 (MAWTHLLLSLLALCTGSVA) ​​for the light chain, or other sequences such as the Campass leader sequence (SEQ ID NO: 945 MGWSCIILFLVATATGVHS) (see US 8,362,208 B2).

[0258] In some embodiments, the nucleic acid may also contain a signal peptide, a short amino acid sequence (13-36 amino acids) at the N-terminus of a secretory protein (such as an immunoglobulin) that mediates translocation of the protein to be secreted through the first membrane of the secretory pathway. This sequence is absent from the mature protein and is cleaved in a co-translational event, mediating the secretion and correct expression of the protein. An appropriate signal sequence can be used to optimize expression of the recombinant protein.

[0259] The present invention also relates to isolated recombinant host cells containing one or more nucleic acid constructs as described above. Host cells useful in the present invention can be prokaryotic, yeast, or higher eukaryotic cells, including, but not limited to, microorganisms such as bacteria (e.g., Escherichia coli, Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell lines infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell lines infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell lines (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter).

[0260] Prokaryotes useful as host cells in the present invention include gram-negative or gram-positive organisms such as Escherichia coli, Bacillus subtilis, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, and Shigella, as well as Bacillus, Pseudomonas, and Streptomyces. One cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. In one embodiment, a method of producing an anti-CD20 antibody described herein is provided, comprising culturing a host cell under conditions suitable for expression of a polynucleotide encoding the antibody and isolating the antibody.

[0261] Nucleic acids encoding antigen-binding domains or antibodies can be used to administer the antigen-binding domains or antibodies to an individual, allowing the encoded protein to be produced in vivo and mediating a therapeutic effect. Polynucleotides can be delivered directly to a subject; for example, a polynucleotide or expression vector can be administered to an individual by directly introducing mRNA or DNA into cells, such as muscle cells. Indirect delivery, in which a polynucleotide is transformed into cells in vitro prior to administration, is also contemplated. Viral vectors, such as defective or attenuated viruses, can also be used.

[0262] In one embodiment, there is provided a method of making an anti-CD3 antigen binding domain or antibody described herein, the method comprising culturing a host cell under conditions suitable for expression of a polynucleotide encoding the antibody and isolating the antibody.

[0263] The present invention also relates to heterologous assays or expression systems comprising canine CD3 and a cell line derived from a different species, for example a human cell line such as HEK.

[0264] This assay involves contacting canine CD3 with a cell line from a different species, e.g., a cell line from a different mammal, e.g., a rodent cell line, or a human cell line such as HEK, e.g., the cell line is transfected with canine CD3 so that it stably or transiently expresses canine CD3.

[0265] kit In another aspect, the invention provides kits for detecting CD3, CD20, and / or CD20 and CD3 for the treatment or prevention of a disease or immune response, e.g., as enumerated herein, and / or for the diagnosis, prognosis, or monitoring of a disease, comprising an antigen-binding domain or antibody of the invention and, optionally, instructions for use. Such kits may include other components, packaging, instructions, or materials to aid in the detection of CD20, CD3, and / or CD20 and CD3 proteins. The kits may include a labeled antigen-binding domain or antibody that binds to CD20, or a binding molecule comprising an antibody that binds to CD20, CD3, and / or CD20 and CD3, and one or more compounds for detecting the label.

[0266] How to generate antibodies The antibodies described herein can be obtained from transgenic mammals, e.g., rodents, that express canine antibodies upon stimulation with CD3 or CD20 antigens. Such rodents are described in WO20018 / 189520 and WO2020 / 074874.

[0267] Thus, the antibodies or fragments described herein can be obtained from mammals, e.g., rodents, e.g., transgenic animals, that express antibodies upon stimulation with canine CD3 or CD20 antigens. Transgenic rodents, e.g., mice, may have a reduced ability to express endogenous antibody genes. Thus, in one embodiment, the rodent has a reduced ability to express endogenous light and / or heavy chain antibody genes. Thus, rodents, e.g., mice, may contain modifications that disrupt the expression of endogenous kappa and lambda light and / or heavy chain antibody genes, such that functional mouse light and / or heavy chains are not produced, e.g., as further described below. Such transgenic rodents have been described in the art and are further described in the Examples below.

[0268] Also included within the scope of the present invention is a method for producing a canine antibody capable of binding to CD3, said method comprising: a) immunizing a transgenic rodent, e.g., a mouse, with a CD3 antigen, wherein the rodent expresses a nucleic acid construct comprising unrearranged canine V, D, and J genes; b) isolating the canine antibody.

[0269] Also included within the scope of the present invention is a method for producing an antibody capable of binding to canine CD3, said method comprising: a) immunizing a transgenic rodent, e.g., a mouse, with a CD3 antigen, wherein the rodent expresses a nucleic acid construct comprising unrearranged canine V, D, and J genes; b) generating a library of sequences comprising heavy and light chain sequences from said rodent, e.g., mouse; c) isolating antibodies comprising heavy and light chain sequences from the library.

[0270] A further step may include identifying antibodies that bind to CD3, for example, by using the functional assays provided in the Examples.

[0271] Methods for preparing or producing the polypeptides, nucleic acids, host cells, products and compositions described herein using in vitro expression libraries include: a) providing a set, collection, or library of nucleic acid sequences encoding amino acid sequences; b) screening said set, collection or library for amino acid sequences capable of binding to / having affinity for CD3; c) isolating the amino acid sequence(s) capable of binding to / having affinity for CD3.

[0272] In the above methods, the set, collection, or library of amino acid sequences can be displayed on a phage, phagemid, ribosome, or suitable microorganism (such as yeast) for, for example, ease of screening. Suitable methods, techniques, and host organisms for displaying and screening (sets, collections, or libraries) of amino acid sequences will be apparent to those skilled in the art (e.g., Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press; 1st edition (October 28, 1996) Brian K. Kay, Jill Winter, John McCafferty). A library, such as a phage library, is generated by isolating cells or tissues expressing antigen-specific antibodies or fragments thereof, cloning sequences encoding the mRNA of the antibodies or fragments thereof obtained from the isolated cells or tissues, and displaying the encoded proteins using the library. The sequences can be expressed in bacteria, yeast, or other expression systems.

[0273] Another aspect also relates to an isolated antibody obtained or obtainable by the above-described method.

[0274] Other Methods and Uses In another aspect, the antibodies, antigen-binding domains, or antigen-binding portions thereof described herein are used for non-therapeutic purposes, such as diagnostic tests and assays. Accordingly, the present invention also relates to a method for detecting canine cells expressing canine CD3 or detecting canine CD3 protein in a biological sample from a canine subject, comprising contacting the biological sample with an antibody, antigen-binding domain, or antigen-binding portion thereof of the present invention, wherein the antibody, antigen-binding domain, or antigen-binding portion thereof is linked to a detectable label. Accordingly, the present invention also relates to a method for detecting canine cells expressing canine CD20 or detecting canine CD20 protein in a biological sample from a canine subject, comprising contacting the biological sample with an antibody, antigen-binding domain, or antigen-binding portion thereof of the present invention, wherein the antibody, antigen-binding domain, or antigen-binding portion thereof is linked to a detectable label. The biological sample may be a biopsy, tissue, blood, serum, plasma, or lymph sample.

[0275] In certain embodiments, the methods can include comparing the amount of binding in a test biological sample with the amount of binding in a control biological sample, where increased binding to the test biological sample relative to the control biological sample can indicate the presence of one or more lymphoma cells in the test biological sample. In some embodiments, the biological sample is dog blood or a needle aspirate. These methods are also provided in in vivo and / or in vitro formats.

[0276] The modification of antibodies for diagnostic purposes is well known in the art. For example, antibodies can be modified with a ligand group such as biotin, or a detectable marker group such as a fluorescent group, a radioisotope, or an enzyme. The compounds of the present invention can be used for diagnostic purposes and can be labeled, for example, using conventional techniques. Suitable detectable labels include, but are not limited to, fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having specific binding partners.

[0277] In another aspect, the antibodies, antigen-binding domains, or antigen-binding portions thereof of the invention that bind to CD20 are used to isolate and / or identify cells that express canine CD20 or cells that contain cell surface proteins that react with these binding agents (e.g., B cells, B lymphoma cells, canine CD20).

[0278] In another aspect, the antibodies, antigen-binding domains, or antigen-binding portions thereof of the invention that bind to CD3 are used to isolate and / or identify cells that express canine CD3 or cells that contain cell surface proteins that react with these binding agents (e.g., B cells, B lymphoma cells, canine CD3).

[0279] The antibodies, antigen-binding domains, or antigen-binding portions thereof described herein can also be used in assays to determine the expression levels of CD20 / CD3, respectively. The expression levels can then be correlated with basal (e.g., control) levels to determine whether a particular disease is present in a patient, the patient's prognosis, or whether a particular treatment regimen is effective.

[0280] Therefore, the present invention relates to the following provisions: Clause 1. A canine antibody, or antigen-binding portion thereof, that binds to canine CD3. Clause 2. The canine antibody or antigen-binding portion thereof of clause 1, which binds to canine CD3 with a monovalent binding dissociation equilibrium constant (KD) of 100 nM to 1000 nM as measured using surface plasmon resonance. Clause 3. The canine antibody, or antigen-binding portion thereof, of clause 1, which binds to canine CD3 with a monovalent binding dissociation equilibrium constant (KD) of less than 100 nM as measured using surface plasmon resonance. Clause 4. A canine antibody or antigen-binding portion thereof according to the preceding clause, which agonistically binds to canine CD3, in particular CD3εδ, and activates said canine T cell receptor. Clause 5. The canine antibody or antigen-binding fragment portion of the preceding clause, wherein said antibody or antigen-binding fragment comprises a complementarity determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 2. Clause 6. The canine antibody or antigen-binding fragment portion of the preceding clause, wherein the antibody or antigen-binding fragment comprises: (a) a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 2; and (b) a CDR of a light chain variable region (LCVR) having an amino acid sequence set forth in Table 2. Clause 7. The canine antibody or antigen-binding fragment portion of the preceding clause, wherein said antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 2. Clause 8. The canine antibody or antigen-binding fragment portion of the preceding clause, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 2; and (b) a light chain variable region (LCVR) having an amino acid sequence set forth in Table 2. Clause 9. The canine antibody or antigen-binding fragment is selected from the group consisting of PMX157, PMX158, PMX160, PMX190, PMX162, PMX163, PMX189, PMX165, PMX167, PMX168, PMX169, PMX170, PMX171, PMX172, PMX173, PMX174, PMX175, PMX176, PMX177, PMX178, PMX179, PMX180, PMX181, PMX182, PMX1 83, PMX184, PMX185, PMX186, PMX187, PMX188, PMX190, PMX192, PMX193, PMX194, PMX195, PMX196, PMX197, PMX198, PMX200, PMX272, PMX273, PMX285 or PMX28, or the CDRs of an HCVR having the amino acid sequence set out in Table 2 for said canine antibody or antigen-binding fragment portion thereof, comprising said HCVR. Clause 10. The canine antibody or antigen-binding portion thereof of the preceding clause, wherein said antigen-binding portion is an scFv, Fv, heavy chain or single domain antibody. Clause 11. The canine antibody, or antigen-binding portion thereof, of the preceding clause, wherein said anti-CD3 antibody comprises one or more heavy chain constant domains. Clause 12. A canine antibody, or antigen-binding portion thereof, that binds to canine CD3 in competition with an antibody, or antigen-binding portion thereof, of the preceding clause. Clause 13. The canine antibody, or antigen-binding portion thereof, of the preceding clause, wherein said antibody, or antigen-binding portion thereof, is conjugated to a therapeutic moiety. Clause 14. The canine antibody, or antigen-binding portion thereof, of Clause 13, wherein said therapeutic moiety is a second antibody, or antigen-binding portion thereof. Clause 15. The canine antibody, or antigen-binding portion thereof, of Clause 14, wherein said second antibody, or antigen-binding portion thereof, binds to a different target. Clause 16. The canine antibody or antigen-binding portion thereof of the preceding clause, wherein said antibody or antigen-binding portion thereof is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle or a radioisotope. Clause 17. A bispecific antibody comprising a first antigen-binding domain that binds to canine CD3 and a second antigen-binding domain that binds to a second target antigen, wherein said first antigen-binding domain comprises the antibody or antigen-binding fragment of any one of clauses 1 to 16. Clause 18. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of clauses 1 to 16, or the bispecific antibody according to clause 17. Clause 19. The antibody or antigen-binding portion thereof of any one of clauses 1 to 16, the bispecific antibody of clause 17, or the pharmaceutical composition of clause 18 for use in the treatment of a disease. Clause 20. A method of treating a disease in a canine subject in need thereof, said method comprising administering an effective amount of the antibody or antigen-binding portion thereof of any one of clauses 1 to 16, the bispecific antibody of clause 17, or the pharmaceutical composition of clause 18.

[0281] Clause 21. The canine antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition of clause 19, or the method of clause 20, wherein the disease is cancer, a B-cell mediated disease, such as a B-cell lymphoma, leukemia, or an immune-mediated disease. Clause 22. The canine antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition of clause 19 or 21, or the method of clause 20 or 21, further comprising separately administering to the subject another therapeutic agent. Clause 23. The canine antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition of clause 22, or the method of clause 22, wherein the therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant, or an immunomodulatory agent such as a cytokine or chemokine. Clause 24. A method for increasing an immune response in a subject, the method comprising administering to said subject the canine antibody or antigen-binding portion thereof of any one of clauses 1 to 16, the bispecific antibody of clause 17, or the pharmaceutical composition of clause 18. Clause 25. A kit comprising the canine antibody or antigen-binding portion thereof of any one of clauses 1 to 16, the bispecific antibody of clause 17, or the pharmaceutical composition of clause 18. Clause 26. The kit of Clause 25, further comprising reagents for detecting the canine antibody or antigen-binding portion thereof. Clause 27. A nucleic acid sequence encoding the antibody or antigen-binding portion thereof of any one of Clauses 1 to 16. Clause 28. The nucleic acid sequence of clause 27, comprising a sequence selected from the SEQ ID NOs set forth in Table 2. Clause 29. A vector comprising a nucleic acid sequence according to any one of clauses 27 and 28. Clause 30. A host cell comprising a nucleic acid sequence according to any one of clauses 27 and 28 or a vector according to clause 29. Clause 31. A method for producing a canine antibody that binds to CD3, said method comprising culturing the isolated host cell of clause 30 and recovering said antibody. Clause 32. A method for producing a canine antibody that binds to CD3, comprising: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a CD3 antigen; b) generating a library of antibodies from said mice; c) isolating antibodies from the library. Clause 33. A method for detecting CD3 protein or an extracellular domain of CD3 protein in a biological sample from a canine subject, comprising contacting the biological sample with the antibody or antigen-binding portion thereof of any one of clauses 1 to 16, wherein the antibody or antigen-binding portion thereof is linked to a detectable label. Clause 34. A combination therapy comprising the antibody or antigen-binding portion thereof of any one of clauses 1 to 16, the bispecific antibody of clause 17, or the pharmaceutical composition of clause 18, and a further therapeutic moiety. Clause 35. The combination therapy of clause 34, wherein said antibody or antigen-binding portion thereof, bispecific antibody, or said pharmaceutical composition and said further therapeutic moiety are administered simultaneously or sequentially. Clause 36. A bispecific canine antigen-binding molecule comprising a first antibody, or antigen-binding portion thereof, that specifically binds to canine CD3, and a second antibody, or antigen-binding portion thereof, that specifically binds to canine CD20. Clause 37. The bispecific canine antigen-binding molecule of clause 36, which binds to human CD3 with a monovalent binding dissociation equilibrium constant (KD) of 100 nM to 1000 nM as measured using surface plasmon resonance. Clause 38. A bispecific canine antigen-binding molecule according to clauses 36 and 37, which provides target-specific cell killing. Clause 39. The bispecific canine antigen-binding molecule of any one of clauses 36 to 38, which induces T cell surface upregulation of CD25 and / or CD69 upon target-mediated cell killing. Clause 40. The bispecific canine antigen-binding molecule of any one of clauses 36 to 39, which activates canine T cells with low IFN-γ secretion in vitro and induces T cell-mediated cytotoxicity of human B cells.

[0282] Clause 41. The bispecific canine antigen-binding molecule of any one of clauses 36 to 40, which induces T cell-mediated cytotoxicity of human B cells. Clause 42. The bispecific canine antigen-binding molecule according to any one of clauses 36 to 41, wherein the first antibody or antigen-binding portion thereof that specifically binds to canine CD3 comprises a heavy chain complementarity determining region (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: set out in Table 2, and a light chain complementarity determining region (LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: set out in Table 2. Clause 43. The bispecific canine antigen-binding molecule of any one of clauses 36 to 42, wherein the second antibody or antigen-binding portion thereof that specifically binds to canine CD20 comprises a heavy chain complementarity determining region (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region (HCVR) comprising a SEQ ID NO: and a light chain complementarity determining region (LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the SEQ ID NOs set forth in Table 2. Clause 44. The bispecific dog antigen-binding molecule of any one of clauses 36 to 43, wherein said second antibody or antigen-binding portion thereof that specifically binds to dog comprises PMX272, PMX285, PMX286, PMX188 or PMX189 as shown in Table 2. Clause 45. The bispecific canine antigen-binding molecule of any one of clauses 36 to 44, wherein said antigen-binding molecule is selected from PMX276, PMX277, PMX278, PMX279, PMX280, PMX281, PMX282, PMX283, PMX284, PMX287, or PMX288. Clause 46. The bispecific canine antigen-binding molecule of any one of clauses 36 to 43, wherein said antigen-binding portion is an scFv, Fv, heavy chain or single domain antibody. Clause 47. The bispecific canine antigen-binding molecule of any one of clauses 36 to 45, wherein said anti-CD3 antibody comprises one or more heavy chain constant domains. Clause 48. The bispecific canine antigen-binding molecule of any one of clauses 36 to 47, wherein said antibody or antigen-binding portion thereof is conjugated to a therapeutic moiety. Clause 49. The bispecific canine antigen-binding molecule of clause 48, wherein said therapeutic moiety is a second antibody or antigen-binding portion thereof. Clause 50. The bispecific canine antigen-binding molecule of clause 49, wherein said second antibody or antigen-binding portion thereof binds to a different target. Clause 51. The bispecific canine antigen-binding molecule of any one of clauses 36 to 50, wherein said bispecific antigen-binding molecule is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope. Clause 52. A pharmaceutical composition comprising the bispecific antigen-binding molecule of any one of clauses 36 to 51. Clause 53. The bispecific canine antigen-binding molecule of any one of clauses 36 to 51 or the pharmaceutical composition of clause 52 for use in the treatment of a disease. Clause 54. A method of treating cancer or a B-cell mediated condition in a canine subject in need thereof, said method comprising administering an effective amount of the bispecific canine antigen-binding molecule of any one of clauses 36 to 51 or the pharmaceutical composition of clause 52. Clause 55. The bispecific canine antigen-binding molecule or pharmaceutical composition according to clause 53 or the method according to clause 54, wherein said disease is cancer, a B-cell mediated disease, such as a B-cell lymphoma, leukemia, or an immune-mediated disease. Clause 56. The bispecific canine antigen-binding molecule of clause 53 or 55, or the pharmaceutical composition of clause 53 or 55, or the method of clause 53 or 55, further comprising separately administering to said subject another therapeutic agent. Clause 57. The bispecific canine antigen-binding molecule of clause 56, the pharmaceutical composition of clause 56 or the method of clause 56, wherein said therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant or an immunomodulatory agent such as a cytokine or chemokine. Clause 58. A method for increasing an immune response in a subject, the method comprising administering to said subject the bispecific canine antigen-binding molecule of any one of clauses 36 to 51 or the pharmaceutical composition of clause 52. Clause 59. A kit comprising the bispecific canine antigen-binding molecule of any one of clauses 36 to 51, or the bispecific antibody or pharmaceutical composition of clause 52. Clause 60. The kit of Clause 59, further comprising reagents for detecting said antibody or antigen-binding portion thereof.

[0283] Clause 61. A nucleic acid sequence encoding the bispecific canine antigen-binding molecule of any one of clauses 36 to 51. Clause 62. The nucleic acid sequence of clause 61, comprising a nucleic acid sequence selected from the SEQ ID NOs set out in Table 2 and / or Table 4. Clause 63. A vector comprising a nucleic acid sequence according to any one of clauses 61 and 62. Clause 64. A host cell comprising a nucleic acid sequence according to any one of clauses 59 and 60 or a vector according to clause 61. Clause 65. A method for producing a bispecific antigen-binding molecule, the method comprising culturing the isolated host cell of clause 64 and recovering said antibody. Clause 66. A method for detecting CD3 protein and CD20 protein in a biological sample from a canine subject, comprising contacting the biological sample with the bispecific antigen-binding molecule of any one of clauses 36 to 51, wherein the antibody or antigen-binding portion thereof is linked to a detectable label. Clause 67. A canine antibody or antigen-binding fragment thereof that binds to canine CD20, in one embodiment said canine antibody or antigen-binding fragment thereof that binds to canine CD20 is selected from the group consisting of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX260, PMX261, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, PMX269, PMX270, PMX271, PMX272, PMX273, PMX274, PMX275, PMX276, PMX277, PMX278, PMX279, PMX280, PMX281, PMX282, PMX283, PMX284, PMX285, PMX286, PMX287, PMX288, PMX289, PMX290, PMX291, PMX292, PMX293, PMX294, PMX295, PMX296, PMX297, PMX298, PMX299, PMX300, PMX301, PMX302, PMX303, PMX304, PMX305, PMX306, PMX307, PMX308, PMX309, PMX310, PMX311, PMX312, PMX313, A canine antibody, or antigen-binding portion thereof, comprising a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 4 for PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to said sequence. Clause 68. The antibody, or antigen-binding portion thereof, comprises an HC variable region sequence comprising an amino acid sequence set forth in Table 4 for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268 or PMX269, or a sequence having at least 75%, 80%, 85%, or 90% sequence identity to said sequence; 68. The canine antibody, or antigen-binding portion thereof, of clause 67, comprising an LC variable region sequence comprising an amino acid sequence as set out in Table 4 for PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268 or PMX269, or a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to said sequence. Clause 69. The canine antibody or antigen-binding portion thereof according to clause 67 or 689, wherein said antigen-binding portion is an scFv, Fv, heavy chain or single domain antibody. Clause 70. The canine antibody, or antigen-binding portion thereof, of any one of clauses 67 to 69, wherein said antibody, or antigen-binding portion thereof, is conjugated to a therapeutic moiety. Clause 71. The canine antibody, or antigen-binding portion thereof, of Clause 70, wherein said therapeutic moiety is a second antibody, or antigen-binding portion thereof. Clause 72. The canine antibody, or antigen-binding portion thereof, of Clause 71, wherein said second antibody, or antigen-binding portion thereof, binds to a different target. Clause 73. The canine antibody or antigen-binding portion thereof of any one of clauses 67 to 72, wherein said antibody or antigen-binding portion thereof is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle or a radioisotope. Clause 74. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of clauses 67 to 73. Clause 75. The canine antibody or antigen-binding portion thereof of any one of clauses 65 to 71, or the pharmaceutical composition of clause 75, for use in the treatment of a disease. Clause 76. A method of treating a B-cell mediated condition in a canine subject in need thereof, said method comprising administering an effective amount of the antibody or antigen-binding portion thereof of any one of clauses 67 to 73 or the pharmaceutical composition of clause 74. Clause 77. The canine antibody or antigen-binding portion thereof or pharmaceutical composition of clause 75, or the method of clause 76, wherein said disease is a B-cell mediated disease, such as a B-cell lymphoma, leukemia, or an immune-mediated disease. Clause 78. The canine antibody or antigen-binding portion thereof or pharmaceutical composition of clause 75 or 77, or the method of clause 76 or 77, further comprising separately administering to said subject another therapeutic agent. Clause 79. The canine antibody or antigen-binding portion thereof or pharmaceutical composition of clause 78, or the method of clause 78, wherein said therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant, or an immunomodulatory agent such as a cytokine or chemokine. Clause 80. A nucleic acid sequence encoding the antibody or antigen-binding portion thereof of any one of clauses 67 to 72.

[0284] Clause 81. The nucleic acid sequence according to clause 80, comprising a sequence selected from the SEQ ID NOs set out in Table 4 for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268 or PMX269. Clause 82. A vector comprising the nucleic acid sequence of any one of clauses 80 and 81. Clause 83. A host cell comprising the nucleic acid sequence of any one of clauses 80 and 81 or the vector of clause 82. Clause 84. A kit comprising the antibody or antigen-binding portion thereof according to any one of clauses 67 to 71, or the pharmaceutical composition according to clause 74. Clause 85. The kit of Clause 84, further comprising reagents for detecting said antibody or antigen-binding portion thereof. Clause 86. A method for producing a canine antibody that binds to CD20 according to any one of clauses 67 to 73, said method comprising culturing the isolated host cell according to clause 81 and recovering said antibody. Clause 87. A method for producing a canine antibody that binds to CD20 according to any one of clauses 67 to 73, comprising: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a CD20 antigen; b) generating a library of antibodies from said mice; c) isolating antibodies from said library. Clause 88. A method for detecting CD20 protein or an extracellular domain of CD20 protein in a biological sample from a canine subject, comprising contacting the biological sample with the antibody or antigen-binding portion thereof of any one of clauses 67 to 73, wherein the antibody or antigen-binding portion thereof is linked to a detectable label. Clause 89. The method of Clause 88, wherein said biological sample is a biopsy, tissue, blood, serum, plasma, or lymph sample. Clause 90. A method of inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of the antibody or antigen-binding portion thereof of any one of clauses 67 to 73, or the pharmaceutical composition of clause 74. Clause 91. A method of killing a tumor cell that expresses CD20, the method comprising contacting said cell with an antibody according to any one of clauses 67 to 73 or a pharmaceutical composition according to clause 74, thereby killing the cell that expresses CD20. Clause 92. The method of Clause 91, wherein said tumor cells are canine tumor cells.

[0285] Therefore, the present invention also relates to the following embodiments: Embodiment 1. A bispecific canine antigen-binding molecule comprising a first antigen-binding domain or an antigen-binding portion thereof that specifically binds to canine CD3, and a second antigen-binding domain that specifically binds to canine CD20. Embodiment 2. The bispecific canine antigen-binding molecule of embodiment 1, which binds to canine CD3 with a monovalent binding dissociation equilibrium constant (KD) of 100 nM to 1000 nM when measured using surface plasmon resonance. Embodiment 3. A bispecific canine antigen-binding molecule according to embodiment 1 or 2, which provides target-specific cell killing. Embodiment 4. The bispecific canine antigen-binding molecule of any one of the preceding embodiments, which induces T cell surface upregulation of CD25 and / or CD69 upon target-mediated cell killing. Embodiment 5. A bispecific canine antigen-binding molecule according to any one of the preceding embodiments, which activates canine T cells with low IFN-γ secretion in vitro and induces T cell-mediated cytotoxicity of human B cells. Embodiment 6. The bispecific canine antigen-binding molecule of any one of the preceding embodiments, which induces T-cell mediated cytotoxicity of human B cells. Embodiment 7. The bispecific canine antigen-binding molecule of any one of the preceding embodiments, wherein the first antigen-binding domain, or antigen-binding portion thereof, that specifically binds to canine CD3 comprises a heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) from a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the SEQ ID NOs: set forth in Table 2, and a light chain complementarity determining region (LCDR1, LCDR2, and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: set forth in Table 2. Embodiment 8. The bispecific canine antigen-binding molecule of any one of the preceding embodiments, wherein said second antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD20 comprises a heavy chain complementarity determining region (HCDR1, HCDR2 and HCDR3) from a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the SEQ ID NOs set forth in Table 4, and a light chain complementarity determining region (LCDR1, LCDR2 and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the SEQ ID NOs set forth in Table 2. Embodiment 9. The bispecific canine antigen-binding molecule of any one of the preceding embodiments, wherein the first and / or second antigen-binding domain or antigen-binding portion thereof comprises a light chain variable region (VL) set forth in PMX272, PMX285, PMX286, PMX188 or PMX189, as shown in Table 2. Embodiment 10. The first and / or second antigen-binding domain or antigen-binding portion thereof comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3; a) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; b) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 480, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 481, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 482; c) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 490, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 491, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 492; d) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 350, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 351, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 352; or e) The bispecific canine antigen-binding molecule of any one of the preceding embodiments, wherein said VL CDR1 comprises the amino acid sequence of SEQ ID NO: 110, said VL CDR2 comprises the amino acid sequence of SEQ ID NO: 111, and said VL CDR3 comprises the amino acid sequence of SEQ ID NO: 112. Embodiment 11. The bispecific canine antigen-binding molecule of any one of the preceding embodiments, wherein said antigen-binding molecule is selected from PMX276, PMX277, PMX278, PMX279, PMX280, PMX281, PMX282, PMX283, PMX284, PMX287, or PMX288.

[0286] Embodiment 12. The molecule comprises: (a) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first heavy chain variable region (VH) comprising a VH complementarity-Determining Region (CDR) 1, a VH CDR2, and a VH CDR3; and (ii) a first light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 68, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 69, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (b) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 87, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 88, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 89, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the second antigen-binding domain or antigen-binding portion thereof comprises a first antigen-binding domain or antigen-binding portion thereof, and a second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462. (c) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 157, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 158, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 159, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (d) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 167, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 168, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 169, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462;

[0287] (e) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 177, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 178, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 179, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (f) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 187, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 188, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 189, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (g) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 327, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 328, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 329, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 480, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 481, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 482; the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 480, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 481, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 482;

[0288] (h) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 337, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 338, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 339, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 490, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 491, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 492; the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 490, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 491, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 492; (i) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 357, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 358, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 359, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (j) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 347, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 348, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 349, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 350, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 351, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 352; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 350, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 351, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 352; or

[0289] (k) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain or antigen-binding portion thereof comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 107, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 108, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 109, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 110, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 111, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 112; 528; and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 110; the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 111; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 112.

[0290] Embodiment 13. The molecule comprises: (a) a first VH comprising the amino acid sequence of SEQ ID NO: 64, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (b) a first VH comprising the amino acid sequence of SEQ ID NO: 84, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (c) a first VH comprising the amino acid sequence of SEQ ID NO: 154, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (d) a first VH comprising the amino acid sequence of SEQ ID NO: 164, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (e) a first VH comprising the amino acid sequence of SEQ ID NO: 174, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (f) a first VH comprising the amino acid sequence of SEQ ID NO: 184, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (g) a first VH comprising the amino acid sequence of SEQ ID NO: 324, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 476; (h) a first VH comprising the amino acid sequence of SEQ ID NO: 334, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 486; (i) a first VH comprising the amino acid sequence of SEQ ID NO: 74, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (j) a first VH comprising the amino acid sequence of SEQ ID NO: 344, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL comprising the amino acid sequence of SEQ ID NO: 346; or (k) the bispecific canine antigen-binding molecule of any one of the preceding embodiments, comprising a first VH comprising the amino acid sequence of SEQ ID NO: 104, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 106. Embodiment 14. The bispecific canine antigen-binding molecule of any one of the preceding embodiments, wherein said antigen-binding domain or antigen-binding portion thereof is an scFv, Fv, heavy chain, or single-domain antibody.

[0291] Embodiment 15. The bispecific canine antigen-binding molecule of any one of the preceding embodiments, wherein said anti-CD3 antigen-binding domain comprises one or more heavy chain constant domains. Embodiment 16 The bispecific canine antigen-binding molecule of any one of the preceding embodiments, wherein said antigen-binding domain or antigen-binding portion thereof is conjugated to a therapeutic moiety. Embodiment 17. The bispecific canine antigen-binding molecule of embodiment 16, wherein the therapeutic moiety is the second antigen-binding domain. Embodiment 18. The bispecific canine antigen-binding molecule of embodiment 17, wherein said second antigen-binding domain binds to a tumor antigen. Embodiment 19. The bispecific canine antigen-binding molecule of any one of the preceding embodiments, wherein said bispecific antigen-binding molecule is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope. Embodiment 20. A pharmaceutical composition comprising a bispecific antigen-binding molecule according to any one of the preceding embodiments. Embodiment 21. A bispecific canine antigen-binding molecule according to any one of Embodiments 1 to 19 or a pharmaceutical composition according to Embodiment 20 for use in treating a disease. Embodiment 22. A method for treating cancer or a B-cell mediated condition in a canine subject in need thereof, comprising administering an effective amount of a bispecific canine antigen-binding molecule of any one of embodiments 1 to 19 or the pharmaceutical composition of embodiment 20. Embodiment 23. The bispecific canine antigen-binding molecule or pharmaceutical composition of embodiment 21, or the method of embodiment 22, wherein the disease is cancer, a B-cell mediated disease, such as a B-cell lymphoma, leukemia, or an immune-mediated disease. Embodiment 24. The bispecific canine antigen-binding molecule of embodiment 21 or 23, or the pharmaceutical composition of embodiment 21 or 23, or the method of embodiment 22 or 23, further comprising separately administering to the subject another therapeutic agent. Embodiment 25. The bispecific canine antigen-binding molecule of embodiment 24, the pharmaceutical composition of embodiment 24, or the method of embodiment 24, wherein the therapeutic agent is a cytotoxic agent, a radiotoxic agent, an immunosuppressant, an immunomodulatory agent such as a cytokine or chemokine, or an antibody or antigen-binding portion thereof that binds to canine CD20. Embodiment 26. A method for increasing an immune response in a subject, comprising administering to the subject a bispecific canine antigen-binding molecule of any one of Embodiments 1 to 19 or a pharmaceutical composition of Embodiment 20. Embodiment 27. A kit comprising the bispecific canine antigen-binding molecule of any one of Embodiments 1 to 19, or the bispecific antibody or pharmaceutical composition of Embodiment 20. Embodiment 28. The kit of embodiment 27, further comprising reagents for detecting the antibody or antigen-binding portion thereof. Embodiment 29. A nucleic acid sequence encoding the bispecific canine antigen-binding molecule of any one of embodiments 1 to 19. Embodiment 30. The nucleic acid sequence of embodiment 29, comprising a nucleic acid sequence selected from the SEQ ID NOs shown in Table 2 and / or Table 4.

[0292] Embodiment 31. A vector comprising the nucleic acid sequence of embodiments 29 and 30. Embodiment 32. A host cell comprising a nucleic acid sequence according to embodiments 29 and 30, or a vector according to embodiment 30. Embodiment 33. A method for producing a bispecific antigen-binding molecule, comprising culturing the isolated host cell of embodiment 32 and recovering the antibody. Embodiment 34. A method for detecting CD3 protein and CD20 protein in a biological sample from a canine subject, comprising contacting the biological sample with the bispecific antigen-binding molecule of any one of embodiments 1 to 19, wherein the antigen-binding molecule is linked to a detectable label. Embodiment 35. A combination therapy comprising the bispecific canine antigen-binding molecule of any one of Embodiments 1 to 19 and an antibody, or antigen-binding portion thereof, that binds to canine CD20. Embodiment 36. A canine antibody or antigen-binding portion thereof that binds to canine CD20, wherein the antibody is selected from the group consisting of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PM A canine antibody or antigen-binding portion thereof, comprising a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 4 for X258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to said sequence. Embodiment 37. The antibody, or antigen-binding portion thereof, comprises an HC variable region sequence comprising an amino acid sequence set forth in Table 4 for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or a sequence having at least 75%, 80%, 85%, or 90% sequence identity thereto; and 37. The canine antibody or antigen-binding portion thereof of embodiment 36, comprising: an LC variable region sequence comprising an amino acid sequence as set forth in Table 4 for PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to said sequence. Embodiment 38. The canine antibody or antigen-binding portion thereof of embodiment 36 or 37, wherein the antigen-binding portion is an scFv, Fv, heavy chain, or single domain antibody. Embodiment 39. The canine antibody or antigen-binding portion thereof of any one of Embodiments 36 to 38, wherein the antibody or antigen-binding portion thereof is conjugated to a therapeutic moiety. Embodiment 40. The canine antibody or antigen-binding portion thereof of embodiment 39, wherein the therapeutic moiety is a second antibody or antigen-binding portion thereof. Embodiment 41 The canine antibody or antigen-binding portion thereof of embodiment 40, wherein the second antibody or antigen-binding portion thereof binds to a tumor antigen. Embodiment 42. The canine antibody or antigen-binding portion thereof of any one of Embodiments 36 to 41, wherein the antibody or antigen-binding portion thereof is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope. Embodiment 43. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof according to any one of embodiments 36 to 42. Embodiment 44. The canine antibody or antigen-binding portion thereof of any one of Embodiments 36 to 42, or the pharmaceutical composition of Embodiment 43, for use in treating a disease. Embodiment 45. A method of treating a B-cell mediated condition in a canine subject in need thereof, comprising administering an effective amount of the antibody or antigen-binding portion thereof of any one of embodiments 36 to 42, or the pharmaceutical composition of embodiment 43. Embodiment 46. The canine antibody or antigen-binding portion thereof or pharmaceutical composition of embodiment 44, or the method of embodiment 45, wherein the disease is a B-cell mediated disease, such as a B-cell lymphoma, leukemia, or an immune-mediated disease. Embodiment 47. The canine antibody or antigen-binding portion thereof or pharmaceutical composition of embodiment 44 or 46, or the method of embodiment 45 or 46, further comprising separately administering to the subject another therapeutic agent. Embodiment 48. The canine antibody or antigen-binding portion thereof or pharmaceutical composition of embodiment 47, or the method of embodiment 47, wherein the therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant, or an immunomodulatory agent such as a cytokine or chemokine. Embodiment 49. A nucleic acid sequence encoding the antibody or antigen-binding portion thereof of any one of Embodiments 36 to 42. Embodiment 50. The nucleic acid sequence of embodiment 49, comprising a sequence selected from the SEQ ID NOs set forth in Table 4 for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269.

[0293] Embodiment 51. A vector comprising the nucleic acid sequence of embodiment 49 or 50. Embodiment 52. A host cell comprising a nucleic acid sequence according to any one of embodiments 49 and 50 or a vector according to embodiment 49. Embodiment 53. A kit comprising the antibody or antigen-binding portion thereof according to any one of embodiments 36 to 42, or the pharmaceutical composition according to embodiment 43. Embodiment 54. The kit of embodiment 53, further comprising reagents for detecting the antibody or antigen-binding portion thereof. Embodiment 55. A method for producing a canine antibody that binds to CD20 described in any one of embodiments 36 to 42, comprising culturing the isolated host cell described in embodiment 52 and recovering the antibody. Embodiment 56. A method for producing a canine antibody that binds to CD20 according to any one of embodiments 36 to 42, comprising: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a CD20 antigen; b) generating a library of antibodies from said mice; c) isolating antibodies from the library. Embodiment 57. A method for detecting CD20 protein or an extracellular domain of CD20 protein in a biological sample from a canine subject, comprising contacting the biological sample with the antibody or antigen-binding portion thereof of any one of embodiments 36 to 42, wherein the antibody or antigen-binding portion thereof is linked to a detectable label. Embodiment 58. The method of embodiment 57, wherein the biological sample is a biopsy, tissue, blood, serum, plasma, or lymph sample. Embodiment 59. A method for inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of the antibody or antigen-binding portion thereof described in any one of embodiments 36 to 42, or the pharmaceutical composition described in embodiment 43. Embodiment 60. A method for killing tumor cells that express CD20, comprising contacting the cells with an antibody described in any one of embodiments 36 to 42 or a pharmaceutical composition described in embodiment 43, thereby killing the cells that express CD20. Embodiment 61. The method of embodiment 60, wherein the tumor cells are canine tumor cells. Embodiment 62. A canine antibody, or antigen-binding portion thereof, that binds to canine CD3. Embodiment 63. The canine antibody or antigen-binding portion thereof of embodiment 62, which binds to canine CD3 with a monovalent binding dissociation equilibrium constant (KD) of 100 nM to 1000 nM as measured using surface plasmon resonance. Embodiment 64. The canine antibody or antigen-binding portion thereof of embodiment 62 or 63, which binds to canine CD3 with a monovalent binding dissociation equilibrium constant (KD) of less than 100 nM, as measured using surface plasmon resonance. Embodiment 65. The canine antibody or antigen-binding portion thereof of any one of embodiments 62 and 63, which agonistically binds to canine CD3, in particular CD3εδ, and activates the canine T cell receptor. Embodiment 66. The canine antibody or antigen-binding portion of any one of embodiments 62 to 65, wherein the antibody or antigen-binding fragment comprises a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 2. Embodiment 67. The canine antibody or antigen-binding portion of any one of clauses 62 to 66, wherein the antibody or antigen-binding fragment comprises: (a) a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 2; and (b) a CDR of a light chain variable region (LCVR) having an amino acid sequence set forth in Table 2. Embodiment 68. The canine antibody or antigen-binding portion of any one of embodiments 62 to 67, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) having an amino acid sequence as set forth in Table 2.

[0294] Embodiment 69. The canine antibody or antigen-binding portion of any one of clauses 62 to 68, wherein the antibody or antigen-binding portion comprises: (a) a heavy chain variable region (HCVR) having an amino acid sequence set forth in Table 2; and (b) a light chain variable region (LCVR) having an amino acid sequence set forth in Table 2. Embodiment 70. The antibody or antigen-binding portion is selected from the group consisting of PMX157, PMX158, PMX160, PMX190, PMX162, PMX163, PMX189, PMX165, PMX167, PMX168, PMX169, PMX170, PMX171, PMX172, PMX173, PMX174, PMX175, PMX176, PMX177, PMX178, PMX179, PMX180, PMX181, PMX182, PMX183, PM 70. The canine antibody or antigen-binding portion of any one of embodiments 62 to 69, comprising a CDR of an HCVR having the amino acid sequence shown in Table 2 for X184, PMX185, PMX186, PMX187, PMX188, PMX190, PMX192, PMX193, PMX194, PMX195, PMX196, PMX197, PMX198, PMX200, PMX272, PMX273, PMX285, or PMX286, or said HCVR. Embodiment 71. The antibody or antigen-binding portion comprises: (i) a heavy chain variable region (VH) comprising a VH complementarity-determining region (CDR)1, a VH CDR2, and a VH CDR3; and (ii) a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3; (a) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 47, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 48, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 49, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 50, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 51, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 52; (b) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 87, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 88, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 89, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 90, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 91, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 92; (c) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 127, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 128, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 129, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 130, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 131, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 132; or (d) The canine antibody or antigen-binding portion thereof according to any one of embodiments 62 to 70, wherein the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 167, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 168, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 169, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 170, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 171, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 172.

[0295] Embodiment 72. The molecule comprises: (a) VH comprising the amino acid sequence of SEQ ID NO: 44, and VL comprising the amino acid sequence of SEQ ID NO: 46; (b) VH comprising the amino acid sequence of SEQ ID NO: 84, and VL comprising the amino acid sequence of SEQ ID NO: 86; (c) a VH comprising the amino acid sequence of SEQ ID NO: 124 and a VL comprising the amino acid sequence of SEQ ID NO: 126; or (d) A canine antibody or antigen-binding portion thereof described in any one of embodiments 62 to 71, comprising a VH comprising the amino acid sequence of SEQ ID NO: 164 and a VL comprising the amino acid sequence of SEQ ID NO: 166. Embodiment 73. The canine antibody or antigen-binding portion thereof of any one of embodiments 62 to 72, wherein the antigen-binding portion is an scFv, Fv, heavy chain, or single domain antibody. Embodiment 74. The canine antibody or antigen-binding portion thereof of any one of embodiments 62 to 73, wherein the anti-CD3 antibody comprises one or more heavy chain constant domains. Embodiment 75. A canine antibody, or antigen-binding portion thereof, that binds to canine CD3 in competition with the antibody, or antigen-binding portion thereof, of any one of Embodiments 62 to 75. Embodiment 76. The canine antibody or antigen-binding portion thereof of any one of Embodiments 62 to 75, wherein the antibody or antigen-binding portion thereof is conjugated to a therapeutic moiety. Embodiment 77. The canine antibody or antigen-binding portion thereof of embodiment 76, wherein the therapeutic moiety is a second antibody or antigen-binding portion thereof. Embodiment 78 The canine antibody or antigen-binding portion thereof of embodiment 76, wherein the second antibody or antigen-binding portion thereof binds to a tumor antigen. Embodiment 79. The canine antibody or antigen-binding portion thereof of any one of embodiments 62 to 78, wherein the antibody or antigen-binding portion thereof is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope. Embodiment 80. A bispecific antibody comprising a first antigen-binding domain that binds to canine CD3 and a second antigen-binding domain that binds to a second target antigen, wherein the first antigen-binding domain comprises the antibody or antigen-binding fragment of any one of embodiments 62 to 79. Embodiment 81. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of embodiments 62 to 79, or the bispecific antibody of embodiment 80. Embodiment 82. An antibody or antigen-binding portion thereof according to any one of embodiments 62 to 79, a bispecific antibody according to embodiment 80, or a pharmaceutical composition according to embodiment 81 for use in treating a disease. Embodiment 83. A method for treating a disease in a canine subject in need thereof, comprising administering an effective amount of the antibody or antigen-binding portion thereof of any one of embodiments 62 to 79, the bispecific antibody of embodiment 80, or the pharmaceutical composition of embodiment 81. Embodiment 84. The canine antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition of embodiment 82, or the method of embodiment 83, wherein the disease is cancer, a B-cell mediated disease, such as a B-cell lymphoma, leukemia, or an immune-mediated disease. Embodiment 85. The canine antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition of embodiment 82 or 84, or the method of embodiment 83 or 84, further comprising separately administering to the subject another therapeutic agent. Embodiment 86. The canine antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition of embodiment 85, or the method of embodiment 85, wherein the therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant, or an immunomodulatory agent such as a cytokine or chemokine. Embodiment 87. A method for increasing an immune response in a subject, comprising administering to the subject a canine antibody or antigen-binding portion thereof described in any one of embodiments 62 to 79, a bispecific antibody described in embodiment 80, or a pharmaceutical composition described in embodiment 81. Embodiment 88. A kit comprising the canine antibody or antigen-binding portion thereof of any one of embodiments 62 to 80, the bispecific antibody of embodiment 80, or the pharmaceutical composition of embodiment 81. Embodiment 89 The kit of embodiment 84, further comprising reagents for detecting the canine antibody or antigen-binding portion thereof. Embodiment 90. A nucleic acid sequence encoding the antibody or antigen-binding portion thereof of any one of embodiments 62 to 80.

[0296] Embodiment 91. The nucleic acid sequence of embodiment 90, comprising a sequence selected from the SEQ ID NOs shown in Table 2. Embodiment 92. A vector comprising the nucleic acid sequence of any one of embodiments 90 and 91. Embodiment 93. A host cell comprising a nucleic acid sequence according to any one of embodiments 90 and 91 or a vector according to embodiment 92. Embodiment 94. A method for producing a canine antibody that binds to CD3, comprising culturing the isolated host cell of embodiment 93 and recovering the antibody. Embodiment 95. A method for producing a canine antibody that binds to CD3, comprising: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a CD3 antigen; b) generating a library of antibodies from said mice; c) isolating antibodies from the library. Embodiment 96. A method for detecting CD3 protein or an extracellular domain of CD3 protein in a biological sample from a canine subject, comprising contacting the biological sample with the antibody or antigen-binding portion thereof of any one of embodiments 62 to 80, wherein the antibody or antigen-binding portion thereof is linked to a detectable label. Embodiment 97. A combination therapy comprising the antibody or antigen-binding portion thereof of any one of embodiments 62 to 80, the bispecific antibody of embodiment 80, or the pharmaceutical composition of embodiment 80, and an additional therapeutic moiety. Embodiment 98. The combination therapy of embodiment 97, wherein the antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition and the additional therapeutic moiety are administered simultaneously or sequentially.

[0297] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by those skilled in the art. While the above disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including how to make and use the disclosure and its best mode, the following examples are provided to enable those skilled in the art to further practice the present disclosure. However, those skilled in the art will understand that the details of these examples should not be construed as limiting the invention, and that the scope of the present invention should be understood from the claims appended hereto and their equivalents. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in light of the present disclosure.

[0298] All documents mentioned herein, including references to gene accession numbers, scientific publications, and patent publications, are incorporated herein by reference in their entirety.

[0299] "And / or," as used herein, should be considered a specific disclosure of each of the two particular features or components with or without the other. For example, "A and / or B" should be considered a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein. Unless the context dictates otherwise, the above feature descriptions and definitions are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0300] The present invention is further described in the following non-limiting examples. [Example]

[0301] Example 1: Immunization of Ky9™ mice Antigen preparation 1. Generation of canine CD3εδ- and CD3εγ-expressing mouse embryonic fibroblasts (MEFs) The coding sequence (CDS) DNA and amino acid sequences of canine CD3ε, CD3δ, and CD3γ are shown in Table 1 (SEQ ID NOS: 1-6). MEFs were grown as monolayers on 90 mm round tissue culture plates in DMEM-high glucose (Life Technologies) supplemented with 10% FBS, 1 mM sodium pyruvate (Sigma-Aldrich), 0.5 mM β-mercaptoethanol (Gibco), and 1% MEM non-essential amino acids (Sigma-Aldrich) at 37°C and 5% CO2. To generate CD3εδ- and CD3εγ-expressing MEFs, wild-type MEFs were stably transfected with two mammalian expression vectors, one encoding the canine CD3ε extracellular (ECD) and transmembrane (TM) domains and the other encoding the CD3δ or CD3γ ECD and TM domains, using Lipofectamine LTX and PLUS™ Reagent (ThermoFisher Scientific) according to the manufacturer's recommended instructions. Both constructs contain PiggyBac inverted repeats that mediate transposition when cotransfected with PiggyBac transposase. The CD3ε expression vector contains a puromycin resistance cassette, while the CD3δ and CD3γ expression vectors carry a hygromycin resistance cassette. After double selection with puromycin and hygromycin, cells were stained with an anti-canine CD3 antibody (clone CA17.2A12, BioRad) to confirm surface expression, and high-expressing cells were further enriched by FACS sorting using a BD FACS Aria. Sorted cells were expanded and frozen to generate master and working cell banks, which were used for mouse immunization, serum titering, and candidate screening.

[0302] 2. Generation of Canine CD20-expressing MEFs Cloning of canine CD20 A search of the CAMFAM_3.1 boxer reference genome was performed using the UCSC genome browser. The genomic sequence of CD20 (MS4A1) was downloaded along with the mRNA sequence AB210085.1. This sequence data was used to design primers to allow amplification of CD20 from the cDNA with additional sequence to allow seamless cloning. This allowed for confirmation of the CD20 sequence in canine blood and seamless cloning into the piggyBac cloning vector.

[0303] Isolation of canine CD20 mRNA and generation of cDNA Beagle whole blood was delivered by Envigo RMS (Alconbury, Huntingdon, UK), and PBMCs were isolated using a Ficoll gradient. Briefly, 10 ml of whole blood was diluted with 25 ml of phosphate-buffered saline (PBS), layered over 15 ml of Ficoll-Paque Plus (Sigma-Aldrich), and then centrifuged at 800 rcf for 10 minutes at room temperature with slow acceleration and no brake. The interphase disk was collected in PBS. Total RNA was isolated from PBMCs using a QIAGEN RNeasy Mini Kit (Qiagen, Hilden, Germany) and standard procedures, followed by on-column DNAse digestion. cDNA was generated using the SuperScript™ IV First-Strand Synthesis System according to standard procedures and anchored oligo-dT primers (ThermoFisher, MA, USA).

[0304] To generate canine CD20-expressing MEFs, cells were co-transfected with a vector containing the canine CD20 DNA coding sequence (Table 1, SEQ ID NO: 22) flanked by PiggyBac inverted terminal repeats and a PiggyBac transposase-containing vector using Lipofectamine LTX and PLUS™ Reagent (ThermoFisher Scientific) according to the manufacturer's recommended instructions. Stably transfected cells were selected with puromycin 48 hours later.

[0305] Immunization of Ky9™ mice As substantially described in WO2018 / 189520 and WO2020 / 074874, Ky9™ mice have been genetically engineered to carry canine immunoglobulin heavy chain (IGH) and light chain (IGL) variable (V) region genes, an IGH D region gene, and IGH and IGL J region genes 5' to mouse constant regions. Thus, Ky9™ mice produce chimeric antibodies with canine heavy and light chain variable regions with mouse constant regions. Information about, or nucleic acids containing, the variable regions of such chimeric antibody chains can be used to generate complete canine antibodies, for example, for therapeutic use in dogs. Rodents containing canine DNA can also serve as animal models for understanding disease and testing pharmaceuticals.

[0306] Ky9™ mice were immunized with MEFs expressing either canine CD3εδ or canine CD20. MEFs were thawed one week before immunization and split twice to reach the required number. On the day of injection, MEFs were trypsinized, washed twice with PBS, and counted before resuspending in injection solution. For the prime immunization, each mouse was injected intraperitoneally with 200 μl of resuspended MEFs in PBS mixed with adjuvant. For the boost immunization, the same protocol was used without adjuvant.

[0307] Serum titer determination Ky9™ mice were bled 10 days before the prime immunization and 10 days after each subsequent booster immunization. Serum and red blood cells were separated using a microvette 200 Z-gel tube (Starstedt AG & Co. KG, Germany), and the titer of canine CD3εδ-specific antibody responses was assessed using a BD Accuri C6 flow cytometer (Becton Dickinson, New Jersey, USA) or a Beckman Coulter CytoFLEX S. Post-immunization serum was serially diluted in FACS buffer (PBS + 3% FBS) and diluted to 10. 5 10 wild-type MEFs or HEK cells, or 10 stably expressing canine CD3εδ or canine CD20 MEFs 5The cells were then either added to the same cells or to HEK cells. Mouse antibodies were detected with a 1 / 200 dilution of BB700-conjugated 20 monoclonal antibody (BD OptiBuild™, Becton Dickinson) against isotypes IgG1, IgG2a, and IgG2b, and binding to these cells was compared to pre-immunization sera. Figure 1 illustrates the results obtained with a cohort of Ky9™ mice immunized with MEFs expressing canine CD3εδ after the booster immunization.

[0308] Example 2: Isolation of antibody-producing cells After immunization of Ky9™ mice with MEFs expressing either canine CD3εδ or canine CD20, antibody-producing cells were isolated as follows.

[0309] Tissue isolation Spleens, lymph nodes, and bone marrow were harvested from immunized mice. Splenocytes were prepared by cutting spleens into small pieces and passing them through a 45 μm cell strainer (Falcon) while rinsing with RPMI-1640 (Lonza, Basel, CH) + 10% FBS on ice. A similar process was used to isolate lymphocytes from lymph nodes. Bone marrow was harvested from femurs and tibias by flushing the bone marrow with RPMI-1640 through a 45 μm cell strainer pre-wetted with RPMI-1640 using a 21-gauge needle. All cell types were pelleted at 300 g for 5 min before being used directly for flow sorting or resuspended in FBS + 10% dimethyl sulfoxide (DMSO) and frozen at -150°C.

[0310] Cell classification Antigen-specific cells can be captured by fluorescently labeled virus-like particles (VLPs) or antigen protein probes. VLPs were generated from HEK cells stably transfected with canine CD3εδ (Table 1, SEQ ID NOs: 1 and 2) or canine CD20 (Table 1, SEQ ID NOs: 22 and 24), then transiently transfected with retroviral gag protein and fluorescently labeled MA (gag matrix fragment p15-GFP fusion protein). Gag expression enables VLP budding from the cells, and MA labels the VLP for fluorescent detection. Briefly, human embryonic kidney (HEK) 293 cells were grown as monolayers on 90 mm round tissue culture plates in DMEM / F12 (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich) at 37°C and 5% CO2. To generate HEK cells expressing canine CD3εδ, HEK293 cells were transfected with two mammalian expression vectors: one encoding the canine CD3ε extracellular (ECD) and transmembrane (TM) domains, and the other encoding the CD3δ or CD3γ ECD and TM domains, along with a vector encoding PiggyBac transposase using polyethyleneimine (PEI MAX: 40 kDa, Polysciences Inc., Eppelheim, Germany). 30 μl of PEI MAX (1 mg ml -1 ), 5 μg of cDNA, and 1 ml of DMEM / F12 were incubated at room temperature for 10 minutes and then added dropwise to a 90 mm plate of 70-80% confluent HEK293 cells. The cells were incubated for 2 days before use. To generate HEK cells expressing canine CD20, HEK293 cells were transfected with a vector containing the canine CD20 cDNA sequence (Table 1, SEQ ID NOs: 22 and 24) flanked by PiggyBac inverted terminal repeats and a vector encoding the PiggyBac transposase using polyethylenimine (PEI MAX: 40 kDa, Polysciences Inc., Eppelheim, Germany) as described previously. Forty-eight hours after transfection, stably transfected cells were selected using the appropriate antibiotic for 7-10 days.

[0311] Antigen-specific B cells can also be captured using labeled antigen protein probes. To produce heterodimeric canine CD3εδ-Fc fusion proteins (Table 1, SEQ ID NOS: 7-10), CHO cells were cotransfected with vectors encoding canine CD3ε-hIgG4PE-knob and canine CD3δ-hIgG4PE-hole. The human Fc contains a knob-in-hole (KiH) mutation that promotes heterodimerization (Merchant AM et al. Nat Biotechnol. 1998 July;16(7):677-81). Fc-tagged probes were purified from culture supernatants using Mab Select Protein A resin (Cytiva) or by AKTA using a Mab Select SuRe column (Cytiva). Conjugation of the CD3εδ-Fc fusion protein to Alexa Fluor 647 was performed using an Alexa Fluor 647 antibody labeling kit (Molecular Probes - Invitrogen) according to the manufacturer's protocol. The degree of labeling was measured using a NanoDrop spectrophotometer. Different dilutions of both probes were tested on splenocytes from mice immunized with CD3εδ-expressing MEFs or with an irrelevant immunogen in combination with Gag-GFP VLPs derived from CD3εδ-overexpressing HEK 293 cells, and antigen-specific B cells were identified and sorted by flow cytometry. The optimal dilution that minimized background staining of irrelevant material was then used to identify and sort antigen-specific B cells using a BD FACSAria Fusion cell sorter (BD Biosciences).

[0312] Sorted B cells were prepared using the 10X Genomics Chromium Single Cell Immune Profiling System and V(D)J kit (10X Genomics) according to the manufacturer's instructions. The nucleotide sequences of the expressed antibodies were determined by 600 cycles (2 × 300 cycles) of Illumina MiSeq sequencing or 2 × 150 cycles of Illumina iSeq, Miseq, MiniSeq, NextSeq, Hiseq4000, or Novaseq sequencing. Sequences were analyzed using a custom tool based on pRESTO / Change-O (Yale University) / IgBlast (NCBI, USA) software to identify paired VH and VL sequences. Clonal lineage information was also constructed based on the identity of the heavy chain V, D, and J genes and the light chain V and J genes.

[0313] An example of antibody sequence analysis of sorted antigen-specific single B cells is shown in Figure 5 of WO2015 / 040401, which shows antibody sequences ordered by heavy chain V gene family usage and clustered to generate a presented phylogenetic tree. Candidate clones are selected from these phylogenetic trees. The nucleic acid and amino acid sequences, VH and VL, of anti-CD3 and anti-CD20 candidates, as well as their corresponding CDRs, are provided in Sequence Tables 2 and 4, respectively.

[0314] Example 3: Candidate antibody formats for expression Anti-canine CD3 candidate bispecific format Generation of CD3 arms To screen for agonistic anti-CD3 candidates, we selected a bispecific killing format with one canine CD3 arm and one rituximab arm (Figure 2). To achieve this, the VH DNA sequences of selected anti-canine CD3 candidates were seamlessly cloned into an expression vector upstream of the human IgG4PE constant region (CH1-hinge-CH2-CH3) with hole and protein A mutations using Gibson assembly (Table 1, SEQ ID NOS: 11 and 12). The VL DNA sequences were cloned into an expression vector upstream of the constant region of the human kappa light chain (Table 1, SEQ ID NOS: 13 and 14).

[0315] Generation of CD20 arm (targeting human CD20) The rituximab VH DNA sequence (Table 1, SEQ ID NOs: 17 and 18) was cloned into an expression vector containing the human IgG4PE constant region (CH1-hinge-CH2-CH3) with knob mutations (Table 1, SEQ ID NOs: 15 and 16), while the rituximab VL DNA sequence (Table 1, SEQ ID NOs: 19 and 20) was cloned into an expression vector containing the human kappa light chain constant region (Table 1, SEQ ID NOs: 13 and 14).

[0316] Four expression vectors encoding the CD3 arm heavy and light chains and the rituximab arm heavy and light chains, respectively, were co-transfected at a 1:1:1:1 ratio into a suitable mammalian cell line, such as CHO cells, for production, as described below.

[0317] Monospecific format of anti-canine CD3 candidates The agonistic anti-CD3 VH and VL pair identified by its bispecific killing capacity (Example 4) was seamlessly cloned into an expression vector upstream of the canine IgG-B effector function-deficient Fc (Table 1, SEQ ID NOS: 29 and 30). The VL DNA sequence was cloned into an expression vector upstream of the canine lambda C5 constant region (Table 1, SEQ ID NOS: 13 and 14).

[0318] Format of anti-canine CD20 candidates To screen anti-CD20 candidates, DNA encoding the VH of the anti-canine CD20 candidates was cloned into an expression vector containing the wild-type canine IgG-B constant region sequence (Table 1, SEQ ID NOS: 27 and 28), while the VL DNA sequence was cloned upstream of the canine lambda C5 constant region (Table 1, SEQ ID NOS: 31 and 32). Expression vectors encoding both the anti-CD20 heavy and light chains were co-transfected into a suitable mammalian cell line, such as CHO cells, for production, as described below.

[0319] antibody production For overgrowth production, 6 x 10 6 Selected CHO cells were seeded in 3 ml of culture medium and incubated at 32°C, 5% CO2 with shaking at 200 rpm. 4% HyClone Cell Boost 7a supplement + 0.4% HyClone Cell Boost 7b supplement + 1% glucose was added to the medium on days 1, 4, 7, and 10. Culture supernatants were harvested on day 12, and antibody concentrations were determined for binding to Protein A using surface plasmon resonance (Biacore 8K, Cytiva Life Sciences).

[0320] Example 4: Screening cascade to identify agonistic bispecific CD3 candidates Candidate anti-CD3 sequences in the bispecific format described in Example 3 were screened for binding to canine CD3εδ and CD3εγ and target-specific cell killing to identify suitable agonistic anti-CD3 candidates.

[0321] Binding screening using cells with surface CD3 expression CHO cell supernatants containing bispecific antibodies were diluted to 1, 5, or 10 μg / ml in FACS buffer (PBS containing 3% FBS) and screened for their ability to bind to canine CD3εδ heterodimers on the cell surface. Briefly, HEK293 cells or MEF cells expressing canine CD3εδ or CD3εγ on their cell surface were incubated with 100 μl of FACS buffer containing the candidate antibody for 30 minutes on ice. As a control, the parental cell line (HEK293 cells or MEF cells not expressing canine CD3) was also stained with the same antibody solution. After staining, the cells were washed with 150 μl of FACS buffer and centrifuged at 300×g for 3 minutes. The supernatant was removed, and the cell pellet was resuspended in FACS buffer containing a 1:1000 dilution of a fluorescently labeled secondary antibody recognizing the human Fc region of the test antibody for 30 minutes in the dark. It was then washed with 150 μl of FACS buffer and centrifuged at 300×g for 3 minutes. Cells were resuspended in FACS buffer and flow cytometry was performed using either a Cytoflex (Beckton Dickinson) or Accuri (Beckman Coulter) cytometer, followed by data analysis using FlowJo (Figure 3).

[0322] Secondary SPR-based binding screening using CD3εδ-Fc fusion protein Candidate CD3 bispecific antibodies were also screened for their ability to bind canine CD3εδ-Fc using surface plasmon resonance (SPR) with a Biacore 8K (Cytiva). Canine CD3εδ-Fc was covalently coupled to the surface of a CM5 chip (Cytiva) by amine coupling. CHO cell supernatant was diluted to 66, 33, and 16.5 nM in HBS-EP+ buffer, and a single-cycle kinetic protocol was run on the chip. Data were analyzed using dedicated software (Biacore Insight evaluation software). Examples of the resulting sensorgrams are shown in Figures 4A-4B.

[0323] In vitro cell-based functional assay to identify agonistic CD3 bispecific candidates To assess their capacity for T cell activation and target-mediated lysis, the candidate CD3 bispecific antibodies generated in Example 3 were tested in an in vitro T cell-mediated cell killing assay using a CD20-expressing canine MDCK cell line as target cells and peripheral blood mononuclear cells (PBMCs) as effector cells.

[0324] To generate target cells, the canine cell line MDCK II (ATCC) was stably transfected with piggyBac-based expression constructs encoding either human CD20 (Table 1, SEQ ID NOS: 21 and 23) or canine CD20 (Table 1, SEQ ID NOS: 22 and 24), along with constructs expressing GFP (Table 1, SEQ ID NOS: 25) and the piggyBac transposase vector. Human CD20+GFP-transfected cells were selected for puromycin and blasticidin resistance, and hCD20 was expressed. 高 GFP (top 5%) cells were FACS sorted by staining for hCD20 expression using anti-human CD20 antibody (clone: ​​2H7, BioLegend) and GFP expression using the FITC channel. Canine CD20+GFP transfected cells were selected for puromycin and blasticidin resistance, and dCD20 高 GFP (top 5%) cells were FACS sorted by staining for dCD20 expression using anti-canine CD20 antibody (Invivogen) and GFP expression using the FITC channel.

[0325] To evaluate bispecific killing, canine peripheral blood mononuclear cells (PBMCs, Envigo) were used as a source of effector cells. PBMCs were isolated from freshly drawn whole blood with heparin sodium anticoagulant using Ficoll-Paque plus (Cytiva, GE17-1440-02) density gradient centrifugation. Briefly, canine blood was diluted 1:1 with phosphate-buffered saline (PBS), carefully layered on top of Ficoll-Paque plus, and centrifuged at 800 × g for 20 min at slow acceleration without disruption. The top and middle discs were diluted with PBS and centrifuged at 420 × g for 10 min to collect the PBMCs in the pellet. A second wash with PBS was performed to remove any Ficoll residue. After the second centrifugation, PBMCs were resuspended in culture medium (PBMC medium = RPMI + 10% heat-inactivated fetal bovine serum + 1% penicillin-streptomycin + 1% non-essential amino acids + 1% L-glutamine + 1% sodium pyruvate + 1% HEPES) before use in the bispecific killing assay.

[0326] To set up the bispecific killing assay, 10,000 MDCK II cells expressing hCD20+GFP or dCD20+GFP were co-cultured with PBMCs at an effector:target ratio of 20:1 in a black-walled 96-well plate. The cell-antibody mixture was incubated in a 1:1 mixture of MDCK II medium (DMEM + 1% L-glutamine + 10% fetal bovine serum) and PBMC medium at 37°C and 5% CO2 for 48 hours. Initial screening was performed using an antibody concentration of 1 μg / ml. After 48 hours of incubation, 40 μl of supernatant was collected from each well for cytokine quantification. IFNγ was measured by ELISA (MABtech) following the manufacturer's instructions, diluting the supernatant 1:30 in dilution buffer.

[0327] Bispecific killing was assessed using GFP signal, which is proportional to the number of viable cells and was used as an endpoint measurement of viable cells. GFP signal was measured using a LARIOstar (BMG Labtech). Data were analyzed using MARS (BMG Labtech), and the percentage of killing in the presence of antibody was calculated using Microsoft Excel. Background signal was obtained from a medium-only control and subtracted from the signal obtained from each test sample. The maximum signal (0% killing) was obtained from a sample of cells treated identically except that antibody was omitted. Graphs were plotted using Graph Prism.

[0328] The results of candidate screening for both bispecific killing and IFNγ release are summarized in Figure 5. It is noteworthy that in vitro bispecific killing potency does not correlate with IFNγ release. For example, PMX165 and PMX182 have similar in vitro killing potency, but PMX165-mediated killing releases three-fold less IFNγ than PMX182. This phenomenon has previously been observed with human anti-CD3 bispecific antibodies (Trinklein et al. 2019 mAbs 11(4):639-652). Our data support the hypothesis that the ability of T cells to kill target cells can be decoupled from cytokine release. Cytokine release appears to correlate with TCR activation; the stronger the binding to the agonist TCR, the stronger the cytokine release (Staflin et al. (2020) JCI insight. 5(7):e133757). Cytokine release is one of the major safety considerations for T cell engager bispecific antibodies. Candidates capable of killing and mediating with low IFNγ release were used as the primary criteria for narrowing down the candidates.

[0329] Based on this criteria, a narrow set of anti-CD3 candidates were selected for full characterization. To further characterize the killing potency and IFNγ cytokine release of the candidates, a dose titration of the narrowed candidates was performed in the range of 10 μg / ml to 0.0001 μg / ml, the results of which are shown in Figure 6, and the calculated EC50 for killing potency in each dose-response curve is shown in Table 6.

[0330] Table 6. Related to Figure 5 TIFF2025540777000003.tif87151

[0331] Example 5: CD3 affinity determination of narrowed down CD3 candidates in a bispecific format In humans, high-affinity CD3 agonists correlate with bispecific killing potency in vitro, but in vivo killing potency is not correlated with in vitro killing potency and is independent of CD3 affinity. Meanwhile, the severity of cytokine release positively correlates with CD3 affinity both in vitro and in vivo (Staflin et al. (2020) JCI Insight. 5(7):e133757; Haber et al. (2021) Scientific Reports 11:14397). Therefore, CD3 affinity is used as a selection criterion to identify potentially safer CD3 candidate arms.

[0332] The binding affinities of the narrowed candidates described in Example 3 were evaluated using Biacore 8K (Cytiva). Briefly, a CM5 sensor chip (Cytiva) was docked to the Biacore 8K and equilibrated at room temperature for 30 minutes. Then, running buffer (10 mM HEPES, pH 6, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween 20) was applied to the SPR chip surface. Canine CD3εδ-Fc was diluted in 10 mM acetate buffer (pH 4.5) and immobilized using a standard amine coupling reaction. Antibody dilutions were prepared by diluting the narrowed candidates with running buffer from 600 nM to 7.4 nM (5 concentrations at 1:3 dilution), and reaction rates were evaluated using multi-cycle kinetics (120 s association, 300 s dissociation). Quantification of reaction rates and / or affinity was performed using Biacore Insight according to standard analytical methods. The affinity of the narrowed down candidates for CD3 is shown in FIG.

[0333] Table 7. Related to Figure 7 TIFF2025540777000004.tif84152

[0334] Example 6: Use of monospecific CD3 candidates for ex vivo T cell activation Another application of agonist anti-CD3 antibodies is for the ex vivo activation and expansion of canine T cells, which can be achieved by using monospecific anti-CD3 antibodies alone or in combination with anti-CD28 antibodies and / or other T cell stimulators (e.g., IL-2).

[0335] Although we performed a screen to identify agonistic CD3 arms, producing CD3 candidates in a monospecific format allows for the identification of CD3 variable region sequences that allow for ex vivo activation of canine T cells, as described in the anti-CD3 monospecific format in Example 4. Monospecific CD3 antibodies were produced in CHO cells as described in Example 3 and purified from CHO supernatants using Protein A resin (MabSelect, Cytiva). Purified antibodies were quality controlled by HPLC size exclusion chromatography (H-SEC) and were all >99% monomeric.

[0336] To evaluate the ability of these monospecific CD3 candidates to activate canine T cells, thawed frozen or fresh canine PBMCs isolated as described above were precoated onto cell culture plates and cultured in PBMC medium supplemented with 50 ng / ml canine IL-2 with either 10 μg / ml of candidate monospecific CD3 antibodies alone in PBS or in combination with 5 μg / ml of anti-canine CD28 antibody (clone 1C6, Fisher Scientific) in PBS for 2–3 hours at 37°C or overnight at 4°C. After 72 hours, cells were imaged to determine the presence of T cell clusters (Figure 8A). The concentration of IFNγ in the supernatant was determined at 72 and 96 hours using a canine IFNγ ELISA (Mabtech) (Figure 8B). Cell surface upregulation of CD25 and PD-1 could also be detected, as exemplified by the use of PMX159 in combination with anti-canine CD28 (Figures 8C–8D). Furthermore, proliferation of canine T cells could also be detected by increased Ki67 staining (Figure 8E). Because anti-CD3 candidates were preselected based on TCR agonism in bispecific killing, most of the tested intact canine anti-CD3 mAbs were able to activate canine T cells ex vivo as well as or better than a commercially available anti-canine CD3 antibody (clone CA17.2A12, Biorad), as demonstrated in Figure 8B.

[0337] The agonistic anti-human CD3 antibody OKT3 has been shown to act as a T cell immunosuppressant. It was approved in 1985 for use in the management of organ transplant rejection. Although highly effective in immunomodulation, the severe side effect of cytokine release syndrome due to strong initial T cell activation led to its discontinuation. A second-generation design using an effector function-deficient OKT3 circumvented the problem of high cytokine release, and in 2022, the FDA approved teplizumab for the treatment of patients recently diagnosed with T1D. These pioneering human studies paved the way for anti-canine CD3 therapy to realize its therapeutic potential. The discovery and validation of agonistic anti-CD3 coupled to an effector function-deficient Fc in dogs will enable therapeutic investigation of this class of candidates for canine autoimmune-related conditions, such as canine T1D.

[0338] Example 7: Binding assays of monospecific anti-canine CD20 candidate antibodies To identify anti-CD20 variable region sequences that can be used as monospecific anti-CD20 and are compatible with the CD3 / CD20 bispecific format, the monospecific anti-CD20 antibodies described in Example 3 were screened using a cell-based CD20 binding assay in which CD20 is displayed on the cell surface as a natural confirmation.

[0339] CD20 binding screening was performed on candidate proteins produced from canine CD20 immunization as described in Examples 2 and 3. CHO cell supernatants containing candidate antibodies were diluted to 10 μg / ml in FACS buffer (PBS containing 3% FBS) and screened for their ability to bind to cell surface-expressed canine CD20. Briefly, 1-2×10 5Canine CD20-expressing HEK cells were incubated with candidate mAbs at a fixed concentration of 10 μg / ml for 1 hour at 4°C, followed by 5 μg / ml of FITC-conjugated anti-canine IgG secondary antibody (Bethyl Laboratories) for 1 hour at 4°C. Cells incubated with the anti-canine IgG FITC secondary antibody without the primary anti-canine CD20 antibody or with an isotype control primary antibody served as negative controls. Data were acquired on either a Beckman Coulter CytoFLEX or BD Accuri C6 Plus flow cytometer and analyzed using FlowJo software. PMX227, PMX228, PMX229, PMX230, PMX231, PMX232, PMX234, PMX235, PMX235, PMX237, PMX238, PMX2 39, PMX240, PMX241, PMX242, PMX243, PMX244, PMX245, PMX246, PMX247, PMX248, PMX249, PMX250, PM The results of binding assays of X251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX260, PMX261, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, PMX269, PMX270, and PMX271 are shown in Figure 9. All candidates except PMX250 and PMX251 showed strong binding ability to CD20-expressing cells.

[0340] Example 8: Killing Potential of Monospecific Anti-Canine CD20 Conjugates To demonstrate cytotoxic killing potential, as described in Example 3, the monospecific format CD20 conjugates with canine wild-type IgG-B Fc (with superior effector function) were further investigated for their functional ability to mediate cytotoxicity, namely complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity, as described below, both killing mechanisms responding to the recruitment of native immune effectors, complement and NK cells, respectively, via the superior effector function canine Fc.

[0341] Complement-dependent cytotoxicity (CDC) activity The CLBL-1 canine lymphoma tumor cell line (University of Veterinary Medicine, Vienna, Germany), which naturally expresses canine CD20 (...

Claims

1. A bispecific canine antigen-binding molecule comprising a first antigen-binding domain or an antigen-binding portion thereof that specifically binds to canine CD3 and a second antigen-binding domain that specifically binds to canine CD20.

2. The bispecific canine antigen-binding molecule of claim 1, which binds to canine CD3 with a monovalent binding dissociation equilibrium constant (KD) of 100 nM to 1000 nM when measured using surface plasmon resonance.

3. 3. The bispecific canine antigen-binding molecule of claim 1 or 2, which provides target-specific cell killing.

4. 4. The bispecific canine antigen-binding molecule of claim 1, which induces T cell surface upregulation of CD25 and / or CD69 upon target-mediated cell killing.

5. The bispecific canine antigen-binding molecule according to any one of claims 1 to 4, which activates canine T cells with low IFN-γ secretion in vitro and induces T cell-mediated cytotoxicity of human B cells.

6. The bispecific canine antigen-binding molecule of any one of claims 1 to 5, which induces T cell-mediated cytotoxicity of human B cells.

7. The bispecific canine antigen-binding molecule of any one of claims 1 to 6, wherein the first antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD3 comprises heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) from a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: set forth in Table 2, and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: set forth in Table 2.

8. The bispecific canine antigen-binding molecule of any one of claims 1 to 7, wherein the second antigen-binding domain or antigen-binding portion thereof that specifically binds to canine CD20 comprises heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) from a heavy chain variable region (HCVR) comprising an amino acid sequence selected from the SEQ ID NOs shown in Table 4, and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) from a light chain variable region (LCVR) comprising an amino acid sequence selected from the SEQ ID NOs shown in Table 2.

9. 9. The bispecific canine antigen-binding molecule of claim 1 , wherein the first and / or second antigen-binding domain or antigen-binding portion thereof comprises a light chain variable region (VL) set forth in PMX272, PMX285, PMX286, PMX188 or PMX189 as shown in Table 2.

10. 10. The bispecific canine antigen-binding molecule of any one of claims 1 to 9, wherein the first and / or second antigen-binding domain or antigen-binding portion thereof comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2 and a VL CDR3; a) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; b) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 480, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 481, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 482; c) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 490, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 491, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 492; d) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 350, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 351, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 352; or e) the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 110, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 111, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:

112.

11. The bispecific canine antigen-binding molecule of any one of claims 1 to 10, wherein the antigen-binding molecule is selected from PMX276, PMX277, PMX278, PMX279, PMX280, PMX281, PMX282, PMX283, PMX284, PMX287, or PMX288.

12. 12. The bispecific canine antigen-binding molecule of any one of claims 1 to 11, wherein the molecule comprises: (a) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first heavy chain variable region (VH) comprising a VH complementarity-Determining Region (CDR) 1, a VH CDR2, and a VH CDR3; and (ii) a first light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 67, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 68, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 69, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (b) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 87, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 88, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 89, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (c) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 157, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 158, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 159, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (d) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 167, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 168, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 169, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (e) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 177, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 178, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 179, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (f) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 187, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 188, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 189, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (g) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 327, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 328, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 329, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 480, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 481, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 482; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 480, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 481, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 482; (h) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 337, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 338, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 339, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 490, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 491, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 492; the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 490, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 491, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 492; (i) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 357, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 358, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 359, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 460, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 461, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 462; (j) the first antigen-binding domain, or antigen-binding portion thereof, and the second antigen-binding domain, or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 347, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 348, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 349, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 350, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 351, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 352; the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, comprising: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, and the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 350, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 351, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 352; or (k) the first antigen-binding domain or antigen-binding portion thereof, and the second antigen-binding domain or antigen-binding portion thereof, the first antigen-binding domain, or antigen-binding portion thereof, comprises: (i) a first VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a first VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 107, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 108, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 109, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 110, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 111, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 112; 12. The bispecific canine antigen-binding molecule of claim 1, wherein the second antigen-binding domain or antigen-binding portion thereof comprises: (i) a second VH comprising a VH CDR1, a VH CDR2, and a VH CDR3; and (ii) a second VL comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 527, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 528, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 529, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 110, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 111, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:

112.

13. 13. The bispecific canine antigen-binding molecule of any one of claims 1 to 12, wherein the molecule comprises: (a) a first VH comprising the amino acid sequence of SEQ ID NO: 64, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (b) a first VH comprising the amino acid sequence of SEQ ID NO: 84, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (c) a first VH comprising the amino acid sequence of SEQ ID NO: 154, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (d) a first VH comprising the amino acid sequence of SEQ ID NO: 164, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (e) a first VH comprising the amino acid sequence of SEQ ID NO: 174, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (f) a first VH comprising the amino acid sequence of SEQ ID NO: 184, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (g) a first VH comprising the amino acid sequence of SEQ ID NO: 324, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 476; (h) a first VH comprising the amino acid sequence of SEQ ID NO: 334, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 486; (i) a first VH comprising the amino acid sequence of SEQ ID NO: 74, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 456; (j) a first VH comprising the amino acid sequence of SEQ ID NO: 344, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO: 346; or (k) a first VH comprising the amino acid sequence of SEQ ID NO: 104, a second VH comprising the amino acid sequence of SEQ ID NO: 524, and a first and second VL comprising the amino acid sequence of SEQ ID NO:

106.

14. The bispecific canine antigen-binding molecule of any one of claims 1 to 13, wherein the antigen-binding domain or antigen-binding portion thereof is an scFv, Fv, heavy chain, or single domain antibody.

15. The bispecific canine antigen-binding molecule of any one of claims 1 to 14, wherein the anti-CD3 antigen-binding domain comprises one or more heavy chain constant domains.

16. The bispecific canine antigen-binding molecule of any one of claims 1 to 15, wherein the antigen-binding domain or antigen-binding portion thereof is conjugated to a therapeutic moiety.

17. 17. The bispecific canine antigen-binding molecule of claim 16, wherein the therapeutic moiety is the second antigen-binding domain.

18. 18. The bispecific canine antigen-binding molecule of claim 17, wherein the second antigen-binding domain binds to a tumor antigen.

19. 19. The bispecific canine antigen-binding molecule of any one of claims 1 to 18, wherein the bispecific antigen-binding molecule is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope.

20. A pharmaceutical composition comprising the bispecific antigen-binding molecule of any one of claims 1 to 19.

21. A bispecific canine antigen-binding molecule according to any one of claims 1 to 19 or a pharmaceutical composition according to claim 20 for use in the treatment of a disease.

22. 21. A method for treating cancer or a B-cell mediated condition in a canine subject in need thereof, comprising administering an effective amount of a bispecific canine antigen-binding molecule of any one of claims 1 to 19 or a pharmaceutical composition of claim 20.

23. 23. The bispecific canine antigen-binding molecule or pharmaceutical composition of claim 21, or the method of claim 22, wherein the disease is cancer, a B-cell mediated disease, such as a B-cell lymphoma, leukemia, or an immune-mediated disease.

24. 24. The bispecific canine antigen-binding molecule of claim 21 or 23, or the pharmaceutical composition of claim 21 or 23, or the method of claim 22 or 23, further comprising separately administering to the subject another therapeutic agent.

25. 25. The bispecific canine antigen-binding molecule of claim 24, the pharmaceutical composition of claim 24, or the method of claim 24, wherein the therapeutic agent is a cytotoxic agent, a radiotoxic agent, an immunosuppressant, an immunomodulator such as a cytokine or a chemokine, or an antibody or antigen-binding portion thereof that binds to canine CD20.

26. 21. A method for increasing an immune response in a subject, the method comprising administering to the subject a bispecific canine antigen-binding molecule of any one of claims 1 to 19 or a pharmaceutical composition of claim 20.

27. A kit comprising the bispecific canine antigen-binding molecule of any one of claims 1 to 19, or the bispecific antibody or pharmaceutical composition of claim 20.

28. 28. The kit of claim 27, further comprising a reagent for detecting the antibody or antigen-binding portion thereof.

29. A nucleic acid sequence encoding the bispecific canine antigen-binding molecule of any one of claims 1 to 19.

30. 30. The nucleic acid sequence of claim 29, comprising a nucleic acid sequence selected from the SEQ ID NOs shown in Table 2 and / or Table 4.

31. A vector comprising the nucleic acid sequence of claims 29 and 30.

32. A host cell comprising a nucleic acid sequence according to claims 29 and 30, or a vector according to claim 30.

33. 33. A method for producing a bispecific antigen-binding molecule, comprising culturing the isolated host cell of claim 32 and recovering the antibody.

34. 20. A method for detecting CD3 and CD20 proteins in a biological sample from a canine subject, the method comprising contacting the biological sample with a bispecific antigen-binding molecule of any one of claims 1 to 19, wherein the antigen-binding molecule is linked to a detectable label.

35. A combination therapy comprising the bispecific canine antigen-binding molecule of any one of claims 1 to 19 and an antibody or antigen-binding portion thereof that binds to canine CD20.

36. 1. A canine antibody, or antigen-binding portion thereof, that binds to canine CD20, wherein the antibody is selected from the group consisting of PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258 , PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto.

37. The antibodies or antigen-binding portions thereof include PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX25 HC variable region sequences comprising the amino acid sequences set forth in Table 4 for PMX232, PMX233, PMX234, PMX235, PMX236, PMX237, PMX238, PMX239, PMX240, PMX241, PMX242, PMX243, PMX244, PMX245, PMX246, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or a sequence having at least 75%, 80%, 85%, or 90% sequence identity thereto; 233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX 250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX 37. The canine antibody, or antigen-binding portion thereof, of claim 36, comprising an LC variable region sequence comprising an amino acid sequence set forth in Table 4 for PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269, or a sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to said sequence.

38. 38. The canine antibody or antigen-binding portion thereof of claim 36 or 37, wherein the antigen-binding portion is an scFv, Fv, heavy chain or single domain antibody.

39. The canine antibody or antigen-binding portion thereof of any one of claims 36 to 38, wherein the antibody or antigen-binding portion thereof is conjugated to a therapeutic moiety.

40. 40. The canine antibody or antigen-binding portion thereof of claim 39, wherein the therapeutic moiety is a second antibody or antigen-binding portion thereof.

41. 41. The canine antibody or antigen-binding portion thereof of claim 40, wherein the second antibody or antigen-binding portion thereof binds to a tumor antigen.

42. 42. The canine antibody or antigen-binding portion thereof of any one of claims 36 to 41, wherein the antibody or antigen-binding portion thereof is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope.

43. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 36 to 42.

44. A canine antibody or antigen-binding portion thereof according to any one of claims 36 to 42, or a pharmaceutical composition according to claim 43, for use in the treatment of a disease.

45. A method for treating a B-cell mediated condition in a canine subject in need thereof, comprising administering an effective amount of the antibody or antigen-binding molecule thereof described in any one of claims 36 to 42, or the pharmaceutical composition described in claim 43.

46. The canine antibody or antigen-binding molecule thereof or pharmaceutical composition of claim 44, or the method of claim 45, wherein the disease is a B-cell mediated disease, for example, a B-cell lymphoma, leukemia, or an immune-mediated disease.

47. The canine antibody or antigen-binding portion thereof or pharmaceutical composition of claim 44 or 46, or the method of claim 45 or 46, further comprising separately administering to the subject another therapeutic agent.

48. The canine antibody or antigen-binding portion thereof or pharmaceutical composition of claim 47, or the method of claim 47, wherein the therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant, or an immunomodulator such as a cytokine or chemokine.

49. A nucleic acid sequence encoding the antibody or antigen-binding portion thereof of any one of claims 36 to 42.

50. 50. The nucleic acid sequence of claim 49, comprising a sequence selected from the SEQ ID NOs set forth in Table 4 for PMX232, PMX233, PMX234, PMX235, PMX237, PMX241, PMX243, PMX244, PMX245, PMX247, PMX248, PMX249, PMX250, PMX251, PMX252, PMX253, PMX254, PMX255, PMX256, PMX257, PMX258, PMX259, PMX262, PMX263, PMX264, PMX265, PMX266, PMX267, PMX268, or PMX269.

51. 51. A vector comprising the nucleic acid sequence of claim 49 or 50.

52. 51. A host cell comprising a nucleic acid sequence according to any one of claims 49 and 50 or a vector according to claim 49.

53. A kit comprising the antibody or antigen-binding portion thereof of any one of claims 36 to 42, or the pharmaceutical composition of claim 43.

54. 54. The kit of claim 53, further comprising a reagent for detecting the antibody or antigen-binding portion thereof.

55. 52. A method for producing a canine antibody that binds to CD20 according to any one of claims 36 to 42, said method comprising culturing the isolated host cell according to claim 52 and recovering said antibody.

56. A method for producing a canine antibody that binds to CD20 according to any one of claims 36 to 42, comprising: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a CD20 antigen; b) generating a library of antibodies from said mice; c) isolating antibodies from said library.

57. 43. A method for detecting CD20 protein or an extracellular domain of CD20 protein in a biological sample from a canine subject, the method comprising contacting the biological sample with an antibody, or antigen-binding portion thereof, of any one of claims 36 to 42, wherein the antibody, or antigen-binding portion thereof, is linked to a detectable label.

58. 58. The method of claim 57, wherein the biological sample is a biopsy, tissue, blood, serum, plasma, or lymph sample.

59. 43. A method for inhibiting tumor growth or metastasis, comprising contacting tumor cells with an effective amount of the antibody or antigen-binding portion thereof of any one of claims 36 to 42, or the pharmaceutical composition of claim 43.

60. 42. A method of killing a tumor cell that expresses CD20, the method comprising contacting the cell with an antibody of any one of claims 36 to 42 or a pharmaceutical composition of claim 43, thereby killing the cell that expresses CD20.

61. 61. The method of claim 60, wherein the tumor cells are canine tumor cells.

62. A canine antibody, or antigen-binding portion thereof, that binds to canine CD3.

63. The canine antibody or antigen-binding portion thereof of claim 62, which binds to canine CD3 with a monovalent binding dissociation equilibrium constant (KD) of 100 nM to 1000 nM when measured using surface plasmon resonance.

64. 64. The canine antibody or antigen-binding portion thereof of claim 62 or 63, which binds to canine CD3 with a monovalent binding dissociation equilibrium constant (KD) of less than 100 nM as measured using surface plasmon resonance.

65. 64. The canine antibody or antigen-binding portion thereof of claim 62 or 63, which agonistically binds to canine CD3, particularly CD3εδ, and activates the canine T cell receptor.

66. 66. The canine antibody or antigen-binding portion of any one of claims 62 to 65, wherein the antibody or antigen-binding fragment comprises a complementarity-determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence as shown in Table 2.

67. 67. The canine antibody or antigen-binding portion of any one of claims 62 to 66, wherein the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) complementarity determining region (CDR) having an amino acid sequence set forth in Table 2; and (b) a light chain variable region (LCVR) CDR having an amino acid sequence set forth in Table 2.

68. 68. The canine antibody or antigen-binding portion of any one of claims 62 to 67, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (HCVR) having the amino acid sequence shown in Table 2.

69. 69. The canine antibody or antigen-binding portion of any one of claims 62-68, wherein the antibody or antigen-binding portion comprises: (a) a heavy chain variable region (HCVR) having the amino acid sequence set forth in Table 2; and (b) a light chain variable region (LCVR) having the amino acid sequence set forth in Table 2.

70. The antibodies or antigen-binding portions thereof include PMX157, PMX158, PMX160, PMX190, PMX162, PMX163, PMX189, PMX165, PMX167, PMX168, PMX169, PMX170, PMX171, PMX172, PMX173, PMX174, PMX175, PMX176, PMX177, PMX178, PMX179, PMX180, PMX181, PMX182, PMX183, PMX184, PMX185, PMX186, PMX187, PMX188, PMX189, PMX190, PMX191, PMX192, PMX193, PMX194, PMX195, PMX196, PMX197, PMX198, PMX19 ... 4, PMX185, PMX186, PMX187, PMX188, PMX190, PMX192, PMX193, PMX194, PMX195, PMX196, PMX197, PMX198, PMX200, PMX272, PMX273, PMX285, or PMX286. The canine antibody or antigen-binding molecule of any one of claims 62 to 69, comprising the CDRs of an HCVR having the amino acid sequence shown in Table 2 for PMX185, PMX186, PMX187, PMX188, PMX190, PMX192, PMX193, PMX194, PMX195, PMX196, PMX197, PMX198, PMX200, PMX272, PMX273, PMX285, or PMX286, or the HCVR.

71. the antibody or antigen-binding portion comprises: (i) a heavy chain variable region (VH) comprising a VH complementarity-Determining Region (CDR) 1, a VH CDR2, and a VH CDR3; and (ii) a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3; (a) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 47, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 48, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 49, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 50, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 51, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 52; (b) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 87, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 88, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 89, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 90, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 91, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 92; (c) the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 127, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 128, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 129, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 130, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 131, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO: 132; or (d) The canine antibody or antigen-binding molecule thereof according to any one of claims 62 to 70, wherein the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 167, the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 168, the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 169, the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 170, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 171, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:

172.

72. The molecule is (a) VH comprising the amino acid sequence of SEQ ID NO: 44, VL comprising the amino acid sequence of SEQ ID NO: 46; (b) VH comprising the amino acid sequence of SEQ ID NO: 84, VL comprising the amino acid sequence of SEQ ID NO: 86; (c) a VH comprising the amino acid sequence of SEQ ID NO: 124, a VL comprising the amino acid sequence of SEQ ID NO: 126, or (d) A canine antibody or an antigen-binding portion thereof according to any one of claims 62 to 71, comprising a VH comprising the amino acid sequence of SEQ ID NO: 164 and a VL comprising the amino acid sequence of SEQ ID NO:

166.

73. The canine antibody or antigen-binding portion thereof of any one of claims 62 to 72, wherein the antigen-binding portion is an scFv, Fv, heavy chain, or single domain antibody.

74. 74. The canine antibody or antigen-binding portion thereof of any one of claims 62 to 73, wherein the anti-CD3 antibody comprises one or more heavy chain constant domains.

75. A canine antibody, or antigen-binding portion thereof, that binds to canine CD3 in competition with the antibody, or antigen-binding portion thereof, of any one of claims 62 to 75.

76. 76. The canine antibody, or antigen-binding portion thereof, of any one of claims 62 to 75, wherein the antibody, or antigen-binding portion thereof, is conjugated to a therapeutic moiety.

77. 77. The canine antibody or antigen-binding portion thereof of claim 76, wherein the therapeutic moiety is a second antibody or antigen-binding portion thereof.

78. 77. The canine antibody or antigen-binding portion thereof of claim 76, wherein the second antibody or antigen-binding portion thereof binds to a tumor antigen.

79. 79. The canine antibody or antigen-binding portion thereof of any one of claims 62 to 78, wherein the antibody or antigen-binding portion thereof is conjugated to a further moiety selected from a half-life extending moiety, a label, a cytotoxin, a liposome, a nanoparticle, or a radioisotope.

80. 80. A bispecific antibody comprising a first antigen-binding domain that binds to canine CD3 and a second antigen-binding domain that binds to a second target antigen, wherein the first antigen-binding domain comprises the antibody or antigen-binding fragment of any one of claims 62 to 79.

81. 81. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 62 to 79, or the bispecific antibody of claim 80.

82. 82. The antibody or antigen-binding portion thereof of any one of claims 62 to 79, the bispecific antibody of claim 80, or the pharmaceutical composition of claim 81 for use in the treatment of a disease.

83. 82. A method of treating a disease in a canine subject in need thereof, the method comprising administering an effective amount of the antibody or antigen-binding portion thereof of any one of claims 62 to 79, the bispecific antibody of claim 80, or the pharmaceutical composition of claim 81.

84. The canine antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition of claim 82, or the method of claim 83, wherein the disease is cancer, a B-cell mediated disease, such as a B-cell lymphoma, leukemia, or an immune-mediated disease.

85. The canine antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition of claim 82 or 84, or the method of claim 83 or 84, further comprising separately administering to the subject another therapeutic agent.

86. The canine antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition of claim 85, or the method of claim 85, wherein the therapeutic agent is a cytotoxic or radiotoxic agent, an immunosuppressant, or an immunomodulatory agent such as a cytokine or chemokine.

87. 82. A method for increasing an immune response in a subject, the method comprising administering to the subject the canine antibody or antigen-binding portion thereof of any one of claims 62 to 79, the bispecific antibody of claim 80, or the pharmaceutical composition of claim 81.

88. 82. A kit comprising the canine antibody or antigen-binding portion thereof of any one of claims 62 to 80, the bispecific antibody of claim 80, or the pharmaceutical composition of claim 81.

89. 85. The kit of claim 84, further comprising a reagent for detecting the canine antibody or antigen-binding portion thereof.

90. A nucleic acid sequence encoding the antibody or antigen-binding portion thereof of any one of claims 62 to 80.

91. 91. The nucleic acid sequence of claim 90, comprising a sequence selected from the SEQ ID NOs shown in Table 2.

92. 92. A vector comprising the nucleic acid sequence of any one of claims 90 and 91.

93. 93. A host cell comprising a nucleic acid sequence according to any one of claims 90 and 91 or a vector according to claim 92.

94. 94. A method for producing a canine antibody that binds to CD3, the method comprising culturing the isolated host cell of claim 93 and recovering the antibody.

95. 1. A method for producing a canine antibody that binds to CD3, comprising: a) immunizing a transgenic mouse expressing a nucleic acid construct comprising a canine heavy chain V gene and a canine light chain V gene with a CD3 antigen; b) generating a library of antibodies from said mice; c) isolating antibodies from said library.

96. 81. A method for detecting CD3 protein or an extracellular domain of CD3 protein in a biological sample from a canine subject, the method comprising contacting the biological sample with an antibody, or antigen-binding portion thereof, of any one of claims 62 to 80, wherein the antibody, or antigen-binding portion thereof, is linked to a detectable label.

97. 81. A combination therapy comprising the antibody or antigen-binding portion thereof of any one of claims 62 to 80, the bispecific antibody of claim 80, or the pharmaceutical composition of claim 80, and a further therapeutic moiety.

98. 98. The combination therapy of claim 97, wherein the antibody or antigen-binding portion thereof, bispecific antibody, or pharmaceutical composition and the additional therapeutic moiety are administered simultaneously or sequentially.

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